Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author.

The dose related effects of phenylbutazone and a methylprednisolone

acetate formulation (Depo-Medrol®) on cultured explants of equine

carpal articular cartilage.

A thesis presented in partial fulfilment of the requirements

for the degree

of Master of Veterinary Science

in Veterinary Pharmacology and Toxicology

at Massey University.

William Thomas Jolly

1996

11 ABSTRACT

Experimental methods involving the mainte�ance of explants of equine articular

cartilage in tissue culture, an amino sugar assay, radiolabelling, and histology were

developed and validated.

The dose related effects of phenylbutazone and Depo-Medrol® on chondrocyte viability

and chondrocyte mediated synthesis and depletion of proteoglycans were investigated

using cultured explants of equine middle carpal joint articular cartilage. Explants from

1 2 horses (941 x 3 mm diameter) were cultured for a total of 5 days, which included

3 days exposure to either phenylbutazone (0, 2, 20, 200, 2000 J.Lg mL-1), or Depo­

Medrol (0, 20, 200, or 2000 J.Lg mL-1). For each explant, amino sugar content was used

as a measure of proteoglycan content, 35S incorporation as a measure of the rate of

proteoglycan synthesis, and the number of pyknotic nuclei as a measure of cell death.

During culture, control explants remained metabolically active and viable but suffered

a net loss of proteoglycans. Proteoglycan loss was reduced by the presence of either

phenylbutazone or Depo-Medrol. This effect was significant at clinically relevant

concentrations of phenylbutazone (2-20 J.Lg mL-1), but not Depo-Medrol (20-200 J.Lg mL·

1). Depo-Medrol caused a dose-dependent suppression of proteoglycan synthesis at all

concentrations, but chondrocyte viability was affected at only the 2000 J.Lg mL-1 dose.

Phenylbutazone affected proteoglycan synthesis and cell viability at only the 2000 J.Lg

mL-1 concentration. At all concentrations, the anti-catabolic effects of each drug

influenced the proteoglycan content of the explants far more than did any anti-anabolic

or cytotoxic drug effect.

The results suggest that the therapeutic potential of both phenylbutazone and Depo­

Medrol may not be just restricted to their anti-inflammatory effects on the soft tissues

of the joint, but may also involve a suppression of the synthesis and/or activation of

proteolytic enzymes within the cartilage itself.

lll PREFACE

Lameness has been reported as the nwrlber one cause of lost training days and failure

to race in the thoroughbred industry (Jeffcott et al., 1 982; Rossdale et al., 1 985). Joint

associated lameness accounted for a third of the lamenesses localised. Causes of joint

lameness include soft tissue inflammations, infections, osteochondritis disicans,

degenerative joint disease, ligamenta! problems and intra-articular fractures. All of the

above conditions may progress to degenerative joint disease.

Corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used

to treat degenerative joint disease (osteoarthritis) in animals and man. Degenerative

joint disease is characterised by deterioration of the articular cartilage, accompanied by

changes to the bone and soft tissues of the joint (Mcllwraith, 1 982). Treatment aims

include resolution of initiating causes, restoration of function, and prevention of further

articular cartilage damage (Mcllwraith & Vachon, 1 988). Because the reparative

response of articular cartilage is inadequate (Desjardins & Hurtig, 1 990), loss of

articular cartilage often limits the complete restoration of athletic function (Bramlage

et al., 1 988; Richardson & Clark, 1 991 ) .

The pathogenesis of degenerative joint disease is incompletely understood (Mcllwraith

& Vachon, 1 988). However, the release of proteoglycans is recognised as one of the

earliest responses of articular cartilage to injury (Mankin, 1 974; Clyne, 1 987). It has

been proposed that proteoglycan depletion resulting from increased proteoglycan

catabolism may leave the chondrocytes and the collagen structural framework more

susceptible to further mechanical damage and thus perpetuate the cycle of degeneration

(Harris et al., 1 972; Mcllwraith & V an Sickle, 1 98 1 ). The relative significance of

chondrocyte mediated proteoglycan catabolism versus that mediated by enzymes

released from the synoviocytes and migrant leucocytes has not been established (Fell

& Jubb, 1 977; Mcllwraith & Van Sickle, 1 981 ; Martel-Pelletier et al., 1 984; Hurtig,

1 988; Mcllwraith & Vachon, 1 988; May et al., 1 99 1 ) . Cytokine and drug induced

IV suppression of proteoglycan synthesis may also contribute to the proteoglycan depletion

in some osteoarthritic conditions (Palmoski & Brandt, 1 983; MacDonald et al., 1 992;

May et al., 1 992).

Methylprednisolone acetate (MP A) and phenylbutazone (PBZ) are the most common

steroid and non-steroidal anti-inflammatory drugs used for treatment of joint injury in

equine athletes. Their soft tissue mediated clinical effects are well recognised (Higgins

& Lees, 1 984). Whether or not they also confer some degree of chondroprotection is

actively debated (Tobin et al., 1 986; Burkhardt & Ghosh, 1 987; Mcllwraith, 1 989).

Furthermore, there is some evidence to suggest their use may actually potentiate the

progression of joint deterioration (Whitehouse & Bostrum, 1 962; Tobin et al., 1 986;

Chunekamrai et al., 1 989; Trotter et al., 1 99 1 ; Shoemaker et al., 1 992). Both the types

and mechanisms of their effects on articular cartilage are subjects of some conjecture

and much controversy (May et al., 1 987; Mcllwraith & Vachon, 1 988; Saari et al., 1 992).

Relatively few controlled in vivo trials have sought to investigate the effects of MP A

or PBZ on equine articular cartilage. Interpretation of specific drug effects from these

trials has been hindered by their small sample numbers, the types of investigative

procedures performed, and a range of confounding variables. The in vitro maintenance

of tissue allows for a more controlled environment in which the study of specific

interactions can be isolated from confounding variables (Tyler et al., 1 982).

The purpose of this study was to investigate the dose related effects of phenylbutazone,

and a methylprednisolone acetate formulation (Depo-Medrol®)1 , on chondrocyte

viability, and chondrocyte mediated degradation and synthesis of matrix proteoglycans

so as to better understand how these drugs exert their effects in vivo. The fol lowing

three hypotheses were tested with respect to each of these parameters; ( 1 ) the drug is

capable of affecting the parameter, (2) the effect is apparent at clinically relevant

concentrations, and (3) the effect is greater at higher concentrations.

Depo-Medrol, Upjohn Inter-American Corporation.

V ACKNOWLEDGEMENTS

This research was partially funded by a grant from the New Zealand Equine Research

Foundation. The author would also l ike to acknowledge and thank the following

people:

Roger Morris; for his encouragement and pragmatic logistical support in setting up the

Masters programme. Elwyn Firth; for his perspective of the subject, his patience and

support, and for his superior editorial skills in teaching me how to write in the scientific

manner. Ted Whittem; for his sound knowledge of analytical chemistry and friendship.

Malcolm Rice, Helen Hodge, Aline Jolly, and Hugh Mortem; for their technical

assistance, and lastly my father and wife for their unfailing encouragement to finish this

thesis.

- TABLE OF CONTENTS

ABSTRACT

• • • . • . . • . • . . . . . • • . . . . . . . . . • . . . • • • • • • . • • • • • • 11

PREFACE

• • • • • • • • • • • • . . • • • • • • • • . • . . • • • • • . • • • • • • • • • • 111

ACKNOWLEDGEMENTS

• • • • . • • • • • • • • . • • . • . • . . • . . • . • • . . . • • • • • • • • • . • V

LIST OF FIGURES

. • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xlll

LIST OF TABLES

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVlll

INTRODUCTION-

A. Articular cartilage structure and function

1 . The chondrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2 . Collagen 4

3 . The proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

4. Measures of matrix biosynthesis and depletion . . . . . . . . . . . 7

5 . Response to injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1

B. Use of intra-articular corticosteroids in the horse

1 . Anti-inflammatory mechanism of action . . . . . . . . . . . . . . . 14

2 . Manufacturer recommendations . . . . . . . . . . . . . . . . . . . . 1 8

3 . Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9

4. Clinical use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1

5 . Adverse effects on joints . . . . . . . . . . . . . . . . . . . . . . . . . 22

C. Effects of corticosteroids on articular cartilage

1 . Normal articular cartilage . . . . . . . . . . . . . . . . . . . . . . . . 24

(i) Chondrocyte morphology . . . . . . . . . . . . . . . 25

(ii) Matrix biosynthesis and depletion;

in vivo results . . . . . . . . . . . . . . . . . . . . . . . 25

(iii) Matrix biosynthesis and depletion;

in vitro results . . . . . . . . . . . . . . . . . . . . . . . 27

2 . Articular cartilage of arthritic joints . . . . . . . . . . . . . . . . . . 28

3 . Articular cartilage repair . . . . . . . . . . . . . . . . . . . . . . . . . 29

D. Use of-phenylbutazone in the horse

1 . Molecular mechanism of action . . . . . . . . . . . . . . . . . . . . 31

2. Manufacturer recommendations . . . . . . . . . . . . . . . . . . . . 34

3. Pharmacokinetics

(i) Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

(ii) Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

(iii) Metabolism and eliminationn . . . . . . . . . . . . . . . . 37

(iv) Oxyphenbutazone kinetics . . . . . . . . . . . . . . . . . . 38

4. Clinical use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

5 . Adverse effects on joints . . . . . . . . . . . . . . . . . . . . . . . . . 40

E. Effect of phenylbutazone on articular cartilage

• • • • • • • • • • • • • • • • • • • • • • • 0 0 0 0 0 0 0 0 . 0 0 • • • 0 • • • 0 40

F. Summary

• • 0 . 0 . 0 . 0 • • • • • • • • 0 0 . 0 • • • 0 • • • 0 0 0 • • • • • • • • 0 . 0 42

G. Objectives

• 0 0 0 . 0 0 0 0 . 0 0 • • 0 0 0 0 0 0 0 0 • • 0 0 0 0 0 0 . 0 0 0 . 0 0 0 0 • • 0 43

H. Hypotheses

• • 0 • • 0 0 • • • 0 • • • 0 • • • • • • • • • • • • • • • • • • 0 . 0 • • • • • 0 43

MATERIALS AND METHODS

SECTION 1: DEVELOPMENT OF THE MODEL

A. Analytical techniques

1 . Biochemical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

2 . Scintillation counting . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

3. DNA assay . . . . . . . . . . . . . . .... . . .. . . . . . . . . . . . . 64

B. Measurement of proteoglycans in the culture media

1 . Factors affecting the amino sugar assay . . . . . . . . . . . . . . 67

2 . Methods to reduce media and drug interference . . . . . . . . . . 72

3 . Separation of chondroitin sulphate from solutions

containing serum proteins . . . . . . . . . . . . . . . . . . . . . . . . 79

4. Separation of proteoglycans from solutions containing

serum proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

5 . Summary . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . . 93

C. Explant culture characteristics

1 . Variation in the amino sugar content and rate of 35S

incorporation between explants from different sites . . . . . . . 96

2. Viability of chondrocytes in cultured explants of

equine articular cartilage . . . . . . . . . . . . . . . . . . . . . . . . . 99

3. Variation in the biosynthetic rate and total amino sugar

concentration relative to time cultured 106

SECTION 11: USE OF THE MODEL

A. Explant Culture

1 . Source of articular cartilage . . . . . . . . . . . . . . . . . . . . . . 1 1 1

2. Preparation of cartilage samples . . . . . . . . . . . . . . . . . . . 1 1 1

3. Methylprednisolone acetate trial . . . . . . . . . . . . . . . . . . . 1 1 4

4 . Phenylbutazone trial . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 I5

B. Post-culture processing

1 . Explants from the radial carpal bones . . . . . . . . . . . . . . 1 1 5

2. Explants from the third carpal bones . . . . . . . . . . . . . . . . 1 1 6

C. Analytical techniques

I. Amino sugar assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 I6

2. Scintillation counting procedure . . . . . . . . . . . . . . . . . . . 1 1 6

3. Histological evaluation . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 7

D. Statistical methods

I. Explants from the radial carpal bones . . . . . . . . . . . . . . . I1 7

2. Explants from the third carpal bones . . . . . . . . . . . . . . . . II 7

RESULTS

A. Depo-Medrol (MP A) trial

1 . Effect of Depo-Medrol on amino sugar content . . . . . . . . . 1 19

2. Effect of Depo-Medrol on 358 incorporation . . . . . . . . . . . 1 1 9

3. Effect of Depo-Medrol on chondrocyte viability . . . . . . . . 1 22

B. Phenylbutazone (PBZ) trial

1 . Effect of PBZ on amino sugar content . . . . . . . . . . . . . . . 124

2. Effect of PBZ on 358 incorporation . . . . . . . . . . . . . . . . . 124

3 . Effect of PBZ on chondrocyte viability . . . . . . . . . . . . . . 127

C. Histological trial observations

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 29

DISCUSSION

A. Discussion of the methods

B. Discussion of the results

C. Summary and conclusions

APPENDIX

A. Tables

134

140

148

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

B. Statistical analyses

. . . ............... .... .. ..... . . . . . .. ..... 161

BIBBLIOGRAPHY

170

FIGURES

2.1 Comparison of the absorption spectra of glucosamine and

galactosamine (from 5-20 J.Lg) . . . . . . . . . . . . . . . . . . . . . . . . . . 47

2.2 Comparison of the absorption spectra of equivalent amounts of

glucosamine, galactosamine, and chondroitin sulphate . . . . . . . . . 47

2.3 Absorption spectra of digested cartilage . . . . . .. . . . . . . . . . . . . . 49

2.4 Comparison of the standard curves of glucosamine, galactosamine,

and chondroitin sulphate . . . . . . . . .... . . . . . . . . .... . . . . . . 50

2.5 The gradation of colours produced by a range of glucosamine

amounts (2.5-20 J.Lg) after being assayed according to the method

of Gatt & Berman. The cuvette containing the yellow/orange

solution is an example of the sensitivity of the assay to any

variation in acidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

2.6 Stability of the amino sugar assay over time . . . . . . . . . . . . . . . . . 53

2. 7 Custom made cartilage chisel . . . . ... . . . . . . . . . . . . . . . .. . . . ·55

2.8 Cartilage punches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

2.9 Quench curve generated by 14C quenched standards . . . . . . . . . . . . 60

2.10 The effect of different wash procedures on the non-bound 35S

content of explants of equine articular cartilage . . . . . . . . . . . . . . . 62

2.1 1 Standard curve for calf thymus DNA assayed according to the

method of Labarca and Paigen (1980) . . . . . . . . . . . . . . . . . . . . . 66

2.12 Absorption spectra of glucosarnine standards dissolved in water . . . 68

2.13 Absorption spectra of glucosarnine standards dissolved in DMEM . . 68

2.14 Absorption spectra of glucosarnine standards dissolved in DMEM

and assayed after oven digestion in 2N HCl . . . . . . . . . . . . . . . . . 68

2.15 Absorption spectra produced by DMEM, aqueous phenol red,

and a 2 5% aqueous DMEM solution in response to the Gatt & Berman (1966) amino sugar assay (relative to a H20 blank,

A= 0.5) . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . .. . ... 74

2.16 Absorption spectra of chondroitin sulphate standards dissolved

in DMEM following dialysis and oven digestion in 2 N HCl

(A= 2 . 0) .......... . .. . .................... ...... 74

2.17 Absorption spectra of DMEM and gentamicin following dialysis

and oven digestion in 2N HCl (relative to a H20 blank,

A= 2.0) . . . . . . . . . .. . .. . . . . . . . . . . . . . .. . . . . . . .. .. . 77

2.18 Comparison of chondroitin sulphate standards dissolved in water

and DMEM after dialysis and oven digestion . . . . . . . . . . . . . . . 78

2.19 The absorption spectrum produced by a crude papain solution . . . . . 81

2.20 Comparison of the absorption spectrum of glucosarnine, and a

glucosamine + papain combination . . . . . . . . . . . . . . . . . . . . . . . 83

2.21 Effect of papain on the assay of glucosamine standards . . . . . . . . . 84

2.22 Effect of serum protein precipitation on the amino sugar

concentration of chondroitin sulphate standards . . . . . . . . . . . . . . . 87

2.23 Effect of serum protein precipitation and dialysis on the

absorption spectra of aqueous chondroitin sulphate standards . . . . . 88

2.24 Effect of serum protein precipitation and dialysis on the

absorption spectra of chondroitin sulphate standards dissolved

in DMEM .................................. . ..... 88

2.25 Effect of culture duration on the viability of chondrocytes

within explants of equine articular cartilage . . . . . . . . . . . . . . . . 1 02

2.26 Photomicrograph showing cell death at the edge of a cultured

explant of equine carpal articular cartilage . . . . . . . . . . . . . . . . . 1 03

2.27 Photomicrograph showing cell death at the articular edge of a

cultured explant of equine carpal articular cartilage . . . . . . . . . . . 1 04

2.28 Photomicrograph showing cell vacuolation in a cultured explant

of equine carpal articular cartilage . . . . . . . . . . . . . . . . . . . . . . 105

2.29 Effect of culture duration on the amino sugar content of explants

relative to their initial wet weight . . . . . . . . . . . . . . . . . . . . . . . 1 08

2.30 Effect of culture duration on the amino sugar content of explants

relative to their final DNA content . . . . . . . . . . . . . . . . . . . . . . 1 08

2.31 Effect of culture duration on 35S incorporation by explants

relative to their initial wet weight . . . . . . . . . . . . . . . . . . . . . . 109

2.32 Effect of culture duration on 35S incorporation by explants

relative to their final DNA content . . . . . . . . . . . . . . . . . . . . . . 109

3.1 Photograph of an open equine middle carpal joint showing the

sites (top =third carpal bone, bottom = radial carpal bone)

where the strips of articular cartilage were harvested from . . . . . . 112

3.2 Strip of articular cartilage from which explants have been cut . . . . 113

3.3 Explants suspended in chilled DMEM awaiting further

processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

4.1 Effect of Depo-Medrol on the amino sugar content of cultured

explants of equine middle carpal joint articular cartilage . . . . . . . . 120

4.2 Effect of Depo-Medrol on 35S incorporation by cultured explants

of equine middle carpal joint articular cartilage . . . . . . . . . . . . . . 121

4.3 Effect of Depo-Medrol on the viability of chondrocytes within

cultured explants of equine middle carpal joint articular

cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

4.4 Effect of phenylbutazone on the amino sugar content of cultured

explants of equine middle carpal joint articular cartilage . . . . . . . . 125

4.5 Effect of phenylbutazone on 35S incorporation by cultured

explants of equine middle carpal joint articular cartilage . . . . . . . . 126

4.6 Effect of phenylbutazone on the viability of chondrocytes

within cultured explants of equine middle carpal joint

articular cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

4. 7 Photomicrograph of an empty lacunae with the displaced nucleus

sitting adjacent (1 OOOx magnification) . .. . . . . . . . . . . . . . . . . . 130

4.8 Photomicrograph of two dead chondrocytes close to the articular

surface ( 4000x magnification) . . . . . . . . . . . . . . . . . . . . . . . . . 131

4.9 Photomicrograph of a chondrocyte cluster

( 4000x magnification) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

4.10 Comparison of the variation in proteoglycan content, as shown

by alcian blue staining, of two sections cut from non-cultured

explants harvested from the radial carpal bone of the same

horse ( 400x magnification) . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

TABLES

2.1 DNA assay of digested cartilage samples . . . . . . . . .. . . . . . . ... 65

2.2 Chemical composition of Dulbecco's modified Eagle medium

(DMEM) ... .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

2.3 Amino sugar assay absorbances following the addition of TCA

and dialysis (524 run) .. . . . ... . .. . ... . . . . .. . . . . . . . .. . . 90

2.4 Amino sugar assay absorbances following the addition of

NH4S04 and dialysis (524 run) . . .. . . . . . . . . . . . . . . . . . . . . . . 93

2.5 Variability in the amino sugar content (AS) of cultured

explants from different carpal bones (/lg mg-1) • • • • • • • • • _ • • • • • 98

2.6 Variability in the incorporation of 35S by cultured explants

from different carpal bones (dpm mg-1) • • • • • • • • • • • • • • • • • • • • 98

2. 7 Chondrocyte death relative to time cultured (dead/total) . . . . . . . . 101

INTRODUCTION

A. ARTICULAR CARTILAGE STRUCTURE AND FUNCTION

1

Articular cartilage is considered to be a hyperhydrated tissue having a water content of

between 65 and 85% (Maroudas et al. , 1969; Mankin, 1974). It consists of a relatively

sparse population of chondrocytes suspended in an extracellular matrix of collagen fibres

and proteoglycan aggregates. Articular cartilage provides an almost frictionless

articulating interface between adjacent bones while absorbing and distributing

concussive, compressive and shearing forces.

The matrix, which is synthesized by the chondrocytes, is wholly responsible for the

biomechanical properties of the tissue. The structure, composition, and cellularity of

articular cartilage at any one site reflects the types and magnitudes of the biomechanical

forces to which that area is subjected (Bramlage et al., 1988). Thus, differences exist

between different joints, sites within a given joint, between species, with age, and with

injury (Mcllwraith & V an Sickle, 1981; Mcllwraith & Vachon, 1988; Vachon et al.,

1990; Richardson & Clark, 1990; Richardson & Clark, 1991 ) . Loss of matrix, through

enzymatic digestion or inhibition of synthesis, compromises the functional capacity of

articular cartilage and can predispose the joint to degenerative joint disease

(osteoarthritis) . (Mcllwraith & Vachon, 1988; Bramlage et al., 1988).

Much of the information pertaining to the composition of equine articular cartilage has

come either from histochemical studies, or has been inferred from research in other

species. Only two studies have biochemically quantified some of the components of

normal equine articular cartilage. Amino sugar and hydroxyproline concentrations were

assayed as measures of the proteoglycan and collagen contents. Amino sugar

concentrations differed widely between studies, between horses, between joints, and

between sites within a given joint. The calculated average glycosaminoglycan content

(dry weight) for one sampling site common to both studies varied from 7.3% (Vachon

et al., 1990) to 12% (Richardson & Clark, 1990). Collagen content was assessed by the

first of these studies only (55.6% dry matter). Human articular cartilage has been

2

reported to have a dry weight composition of 60% collagen, 30% proteoglycans

(approximately 90% of which are glycosaminoglycans), and 10% glycoproteins, lipids

and chondrocytes (Mankin, 1974).

Histological techniques have been used in many anatomical and comparative studies.

However, some confusion exists over the terminology used to describe the appearance

of chondrocytes and matrix in histological sections. The term lacuna originated purely

as a histological descriptive term and referred to an area which normally contained one

or more chondrocytes surrounded by 'finely textured pericellular matrix' and into which

'fibrous matrix' did not encroach (Stockwell, 1979). Pericellular matrix usually refers

to the matrix immediately surrounding the chondrocyte within the 'rim' of the lacuna,

but is also sometimes used synonymously with territorial matrix. Territorial matrix

describes the broad circular basophilic zone radiating out from many of the

chondrocytes, while the inter-territorial matrix is basically the rest. Chondrones and

chondrocyte clusters are also both histologically descriptive terms used to describe

closely associated groups of chondrocytes which are thought to have resulted from

relatively recent mitotic episodes. In contrast, the term chondron describes a physically

isolatable structure consisting of a felt-like pericellular capsule constraining one or more

chondrocytes and a quantity of pericellular matrix (Poole et al., 1988).

1. The chondrocytes

The chondrocytes synthesize and maintain matrix structure and composition.

Chondrocyte shape, size and density vary with distance from the articular surface,

location within the joint, maturity, and joint pathology (Stockwell, 1979; Oikawa et al. ,

1989; Mcllwraith & V an Sickle, 1981 ). Chondrocyte density and metabolic activity are

greatest in areas of the joint exposed to regular weight bearing (Palmoski et al., 1980).

Four zones are recognised in adult articular cartilage. The tangential zone, or

superficial zone, exists immediately below the articular surface and contains flattened

chondrocytes. Next is the transitional zone which contains randomly distributed

rounder chondrocytes. The third zone is the radiate zone where the chondrocytes have

3

a similar appearance to those in the transitional zone, but are arranged in short irregular

columns. The deepest is the calcified zone, containing chondrocytes surrounded by

hydroxyapatite, and separated from the radiate zone by the tideline (Weiss, 1968; Lust

et al., 1972).

The chondrocytes of the transitional and radiate zones are retained within 'felt-like'

pericellular capsules (Poole et al., 1988). These fibrous capsules can contain one or

several chondrocytes. The capsule, the chondrocytes and the pericellular matrix are

together referred to as a chondron. Frayed collagen fibres radiate from these chondrons

and suggest a shear resistant, structural interrelationship between the capsular

components and the type 11 collagen fibres of the matrix. The pericellular

microenvironrnent contained within the chondron's fibrous capsule is thought to be

involved in the regulation of chondrocyte metabolism (Poole et al. , 1988).

Mankin ( 1974) proposed that the chondrocytes of immature articular cartilage actively

proliferate, but at a rate which dramatically decreases as the animal grows and matures.

The chondrocytes of normal mature cartilage are essentially amitotic. As in other

animals, the articular cartilage of neonatal foals exists as part of an articular-epiphyseal

cartilage complex, which is thick in the neonate and decreases as endochondral

ossification proceeds (Firth & Greydanus, 1987). In a study examining age-related

changes in the thickness of the articular cartilage of equine third metacarpal bones,

Oikawa et al. ( 1989) found there was no apparent histological pattern to the

chondrocyte distribution in foals for their first 6 months of life. However, from

approximately 6 months of age onwards, the more superficial chondrocytes were

arranged parallel to the articular surface, and the middle and deeper chondrocytes

existed in a more perpendicular type of pattern. As the yearlings matured the

demarcation of calcified cartilage from non-calcified cartilage became evident as a

distinct border, referred to as the tideline. At 1 9 months of age tbe chondrocyte

distribution was characterised as being organised into the four classical zones, and after

24 months of age little further change in thickness was seen (Oikawa et al. , 1989).

4

The molecular mechanisms regulating chondrocyte replication are unknown (Tyler,

1 985 ; Hickery et al. , 1990). There are no studies on changes in equine chondrocyte

mitotic rate with age, or on the reasons for normal articular chondrocytes being

essentially amitotic. However, chondrocytes within mature equine cartilage still retain

a limited ability to replicate when exposed to adverse conditions (Riddle, 1 970;

Mcllwraith & Van Sickle, 1 98 1; Glade, 1990).

Adult articular cartilage is avascular, aneural, and devoid of lymphatics. Supply of

nutrients and exchange of metabolic byproducts is by simple diffusion across the matrix

to and from the synovial fluid and subchondral bone vasculature. This is aided by the

movement of the fluid component of articular cartilage caused by the intermittent

compression associated with weight bearing and joint movement (Maroudas & Evans,

1974; Palmoski et al., 1980).

The chondrocytes of the tangential zone contain few intracytoplasmic filaments or

inclusion bodies, whereas the chondrocytes of the transitional and radiate zone contain

greater amounts of mitochondria, rough endoplasmic reticulum and golgi complex,

indicative of greater metabolic activity (Lust et al., 1 972). The metabolic, catabolic,

and biosynthetic activity of each chondrocyte is sensitive to its immediate extracellular

oxygen tension, acidity, metabolite and nutrient concentrations (May et al., 1 99 1 ). The

surrounding matrix composition, the depth from the articular surface, and the

compression loading dynamics are thus all important determinants of the metabolic

functioning of individual chondrocytes (Maroudas et al., 1 969; Maroudas & Evans,

1974; Handley & Lowther, 1 977; Palmoski et al. , 1980; Sandy et al., 1 980; Jones et al.,

1 982; Schneiderman et al., 1986; Gray & Gottlieb, 1 983).

2. Collagen

The collagen fibrils in articular cartilage interact to form a woven mesh restraining the

proteoglycan aggregates, and providing tensile strength and resistance to shearing forces

(Hickery et al., 1 990). Collagen strength depends on the formation of covalent bonds

between its polymerized collagen molecules, but heterotypic cross-linking with other

5

protein types is also important to the stability of the resultant network (Broom, 1 982).

Type 2 collagen fibrils predominate in adult equine articular cartilage (Vachon et al. ,

1 990). The number, size and orientation of these fibrils vary with the depth of the

articular cartilage. Classically (Benninghoff, 1 925) they were described as being closely

packed and arranged parallel to the articular surface superficially, and more widely

spaced and more perpendicular to the articular surface with increasing depth. The

alignment of individual collagen fibrils has now been shown to be somewhat more

random, with distribution surrounding chondrocytes and in the superficial layer being

the most ordered (Stockwell, 1 979; Poole et al. , 1988). However, the resulting complex

structural framework is responsible for restraint of the aggregated proteoglycans, and

reflects the biomechanical conditions prevailing in any one area (Riddle, 1 970; Lust et al. , 1 972; Heinegard et al. , 1 987; Mcllwraith & Vachon, 1 988; Desjardins & Hurtig,

1 990).

3. The proteoglycans

Proteoglycans are high molecular weight molecules (1 000-3000 kDa) consisting of a

central ·protein core (200-300 Kda) to which a large number of glycosaminoglycan side

chains are covalently bound. Most of the proteoglycans contained within the· collagen

framework of articular cartilage are bound non-covalently to strands of hyaluronic acid,

and stabilised with l ink proteins, to form large proteoglycan aggregates (Stockwell,

1 979; Hardingham et al., 1990). However, some are also firmly associated with the

collagen fibrils (Sandy et al., 1 978; Hascal & Kimura, 1 982; Nakano et al. , 1 985). The

physical size and steric conformation of the resulting aggregates are largely responsible

for the ability of the collagen mesh to restrain the proteoglycan content of the matrix.

Up to 90% of the total mass of the proteoglycan aggregates are glycosaminoglycans,

which are carbohydrate polymers ( 4-30 Kda) consisting of repeating disaccharide units.

Glycosaminoglycan concentrations vary between individuals, between different joints,

between sites within joints, with distance from the articular surface, and with the

existence of any joint disease (Jones et al., 1 977; Schneiderman et al., 1 986; Richardson

& Clark, 1 990). Keratansulphate and chondroitin sulphate are the main

6

glycosaminoglycans of articular cartilage. Keratansulphate side chains tend to be

concentrated towards the hyaluronic acid binding end, and chondroitin sulphate side

chains predominate on the peripheral two thirds of the protein core (Hascal & Kimura,

1 982). The relative proportions of keratansulphate and chondroitin sulphate can also

vary between individuals, between joints, �thin joints, and with distance from the

articular surface (Maroudas et al., 1 969; Jones et al., 1 977).

Keratansulphate consists of a repeating galactose and N-acetylglucosamine disaccharide

unit. The glucosamine residue in the keratansulphate molecule is normally sulphated

at the sixth carbon atom, but this sulphate group may be absent. Alternatively an extra

sulphate group may be present at the sixth carbon atom of the galactose residue

(Stockwell, 1 979). Most disaccharide units contribute just one negative charge.

Chondroitin sulphate consists of a repeating glucuronic acid and N-acetylgalactosamine

disaccharide unit. Chondroitin sulphate can exist as either chondroitin-6-sulphate or

chondroitin-4-sulphate, depending on whether the sulphate group is bound to the sixth

carbon atom or the fourth carbon atom of the galactose molecule. The proportion of

chondroitin-6-sulphate increases with age. The length of chondroitin sulphate chains

varies, and may consist of up to 1 00 disaccharide units. Generally each disaccharide

unit is considered to contain one carboxyl and one sulphate group and contribute two ·

negative charges, but there is good evidence to suggest mammalian chondroitin sulphate

molecules are 'undersulphated' at the protein binding end (Wasteson & Lindahl, 1 97 1 ).

In contrast, shark chondroitin sulphate, often used as a reference standard, tend to have

additional sulphate groups along its length and may average more than two negative

charges per disaccharide unit (Stockwell, 1 979).

The sulphate and carboxyl groups on the glycosaminoglycan side-chains result in the

highly polyanionic nature of the matrix. The mutual repulsion of these negative charges

dictates the steric conformation of the proteoglycan aggregates. Furthermore, the

restraint of these highly polyanionic aggregates attracts a variety of cations, and the

consequent large osmotic potential accounts for the high water content of cartilage.

Cartilage is thus considered to be a biphasic material with a fluid phase which is readily

7

interchangeable with components of the synovial fluid. Fluid movement occurs by

simple osmotic diffusion, or when the cartilage is subjected to compression. The

restraint of these aggregated proteoglycans with their associated moveable water

molecules results in the compressive stiffness, load distributing and friction minimising

properties of articular cartilage. The balance between the expansive forces associated

with the proteoglycans and the restraining tension in the collagen network, results in the

compressive stiffness of the articular cartilage (Hardingham et al., 1 990). A change in

either the proteoglycan content or structural integrity of the collagen framework can thus

alter the functional capacity of articular cartilage (Harris et al., 1 972; Glade et al., 1 983;

Mcllwraith & Vachon, 1 988)

Proteoglycan levels in adult articular cartilage are maintained by a continuous synthesis

and turnover by the chondrocytes (Hickery et al., 1 990). In contrast to the rate of

synthesis of collagen, the turnover of proteoglycans is considered to be relatively rapid,

but estimates of the 'normal' rate of turnover vary widely (Maroudas & Evans, 1 974).

The activity of the chondrocyte, whether anabolic or catabolic, is influenced by the

conditions immediately surrounding it. Physicochemical factors such as matrix

hydration, osmotic pressure, pH, mechanical loading, and hormone and/or metabolic

gradients are likely to be important (Schneiderman, et al., 1 986; Poole et al., 1 988).

The rate of glycosaminoglycan synthesis has been shown to vary inversely with local

glycosaminoglycan concentrations (Maroudas & Evans, 1 974; Handley & Lowther,

1 976; Sandy et al., 1 978; Sandy et al., 1 980). Rates of synthesis also vary between

species, individuals, ages, joints, normal and damaged joints, sites within joints, and

with methods of quantification (Maroudas & Evans, 1 974; Sandy et al., 1 980;

Richardson & Clark, 1 991 ).

4. Measures of matrix biosynthesis and depletion

Depletion of proteoglycan content results from imbalance of the normal turnover of

proteoglycans, and can be due to a relative increase in the catabolic rate, or a relative

decrease in the synthetic rate.

8

The rate of 35S incorporation by cultured explants of equine articular cartilage has been

used commonly as a comparative measure of proteoglycan synthesis in in vitro trials.

This method has also been used to infer what the in vivo synthetic rate may have been

immediately prior to removal of the explant (Mankin et al., 1972; Behrens et al., 1976;

Tyler, 1985; Chunekamrai et al., 1989; Fubini et al., 1993). However, the rate of 35S

incorporation can also vary with the S04 2• concentration of the medium, the time each

explant is exposed to the radiolabel, and the timing of the radiolabelling with respect

to the start of the culture (Maroudas & Evans, 1974). Injection of Na/5S04 and

glycine-H3 directly into the joints of live rabbits has also been used to. compare relative

proteoglycan and protein synthetic rates between joints (Behrens et al., 1976; Mankin

& Conger, 1966).

The Bitter & Muir ( 1962) assay for uronic acid has commonly been used to estimate

the glycosaminoglycan content of articular cartilage (Glade et al., 1983; Tyler, 1985;

Trotter et al., 1991 ). This assay estimates the chondroitin sulphate portion of the total

glycosaminoglycan content of articular cartilage only. The relative proportions of

chondroitin sulphate to keratansulphate vary with age, site, depth from the articular

surface, and joint pathology.

Cationic dyes, such as alcian blue and dimethylmethylene blue (DMB), have been used

to quantify released or extracted proteoglycans (Dingle et al., 1979; Mcllwraith & Van

Sickle, 1981; Jubb, 1984; Tyler, 1985). Although these dyes bind to both major types

of glycosaminoglycan present in articular cartilage, the resulting absorbance produced

(525 nm) is much greater for chondroitin sulphate than for keratansulphate. Extraction

techniques produce variable yields of proteoglycans. Furthermore, yield percentages

tend to be affected by actual proteoglycan content (Stockwell, 1979). The DMB method

has been increasingly used in comparison to other assays because of its relative

simplicity, improved sensitivity, and ease of automation. When used in conjunction

with tissue culture systems the DMB method can be subject to interference from several

of the components of either the media or tissue digest (Sabiston et al., 1985; Farndale

et al., 1986; Templeton, 1988). Proteolysis in combination with other purification

techniques can remove much of this interference (Sabiston et al., 1985; Farndale et al.,

9

1986). However, all reference standards should be spiked with similar concentrations

of the interfering components, and be subjected to the same procedures also (Sabiston

et al., 1985). The use of shark chondroitin sulphate as a reference standard may result

in an underestimation of the true amount of mammalian chondroitin sulphate present

(Stockwell, 1979))

Histochemical staining techniques employing safranin-0 and alcian blue have been used

to assess variation in glycosaminoglycan content between histological sections of

articular cartilage (Lust et al., 1972; M ell wraith & V an Sickle, 1981; Hurtig et al.,

1988; Shamis et al., 1989; Chunekamrai et al., 1989). The number of negatively

charged groups associated with a glycosaminoglycan molecule varies along its length

and with its type (Wasteson & Lindahl, 1971). The affinity of these charged groups for

some of the cationic dyes can vary with pH and competition from other cations. Quite

apart from any trial differences, variations may also result from variable proteoglycan

losses during processing of the articular cartilage samples prior to staining. As such,

the resultant staining intensities and the distribution of staining intensities observed may

not be representative of the initial proteoglycan content (Hunziker et al., 1992).

Assays for both individual and total amino sugar content can be used to quantitatively

estimate glycosaminoglycan content also (Mankin, 1974; Glade et al., 1983; Richardson

& Clark, 1990; Vachon et al., 1991). Keratansulphate and chondroitin sulphate have

. equal proportions of amino sugar. Hyaluronic acid and glycoproteins also contribute

to the total assayable amino sugar levels, but their contributions are considered small

in comparison to those coming from keratansulphate and chondroitin sulphate.

Analytical techniques used to estimate either total or individual amino sugar content

include modifications of the Elson & Morgan (1933) calorimetric assay for amino

sugars, gas liquid chromatography methods, and methods involving ion chromatography.

These all require extensive hydrolysis of the proteoglycans to yield isolated amino sugar

molecules before analysis, and hence give no indication of the distribution or size of the

proteoglycans.

5. Response to injury

10

The loss of proteoglycans is recognised as an early response of articular cartilage to

injury. The collagen structural framework is much more susceptible to mechanical

damage if the proteoglycan content is depleted (Mcllwraith & Van Sickle, 1 98 1 ;

Malemud et al., 1 987). Whether damage to the collagen structural framework precedes

or follows proteoglycan depletion, both are considered inextricably linked in the

progressive degeneration of articular cartilage in the horse (Mcllwraith & Vachon,

1 988).

It is not known whether the depletion of proteoglycans found in many cases of

degenerative joint disease involves an increase in chondrocyte mediated catabolism, or

whether the cartilage damage is the result of extrinsic enzymes originating from the

synoviocytes, bacteria, or migrant leucocytes (Fell & Jubb, 1977; Mcllwraith & Van

S ickle, 1 98 1 ; Mcll"WTaith, 1 982; Martel-Pelletier et al., 1 984; Hurtig, 1 988; Mcllwraith

& Vachon, 1 988; May et al., 1 991 ) . Cytokine and drug induced suppression of

synthesis may also be a significant factor in the chronic progression of some

osteoarthritic conditions (Behrens et al., 1 974; Tyler, 1 985; May et al., 1 992).

Chondrocytes have been shown to produce catabolic enzymes in response to a number

of adverse conditions (Sapolsky et al., 1 98 1 ; May et al., 1 991 ). These include physical

trauma, chemical irritants, pH changes, cytokines, inflammatory mediators or

modulators, and inadequate supplies of nutrition. The cytokines and inflammatory

mediators and modulators can originate from the chondrocytes themselves, synoviocytes,

subchondral bone, granulation tissue, or leucocytes which have migrated into the

synovium or synovial fluid (Dingle et al., 1 979; Morales, 1 984; Tyler, 1 985; Hickery

et al., 1 990). Tumour necrosis factor, prostaglandin E2 and interleukin-1 are thought

to be involved in both stimulating increased enzymatic production, and in activating the

intrinsic enzymatic degradation in articular cartilage (Dingle, 1 984; May et al., 1 99 1 ;

Price et al., 1 992; May et al., 1 992). Catalytic amounts of the neutral metalloproteinase

stromelysin, once activated, can also activate at least one other pro-metalloproteinase

found in articular cartilage (Ogata et al., 1 99 1 ).

11

Very little work has been done to investigate the types of enzymes involved in the

catabolism of equine articular proteoglycans. As a consequence, much of the

information concerning the mechanisms involved in equine articular cartilage catabolism

must be extrapolated from work done on other species. However, increased amounts

of the neutral metalloproteinase stromelysin have been shown to be produced by equine

chondrocytes cultured under adverse conditions (May et al., 1991 ).

Human articular cartilage contains a number of neutral and acid metalloproteinases

capable of catabolizing proteoglycans. Much of the metalloproteinase content exists

either in the latent forms or is inhibited by factors within the carti lage (Woessner &

Selzer, 1984; Martel-Pelletier et al., 1985; Flannery et al., 1992). Two studies have

reported higher active concentrations of neutral and acid metalloproteases in

osteoarthritic cartilage, but in both cases the differences were not statistically significant

(Martel-Pelletier et al., 1984; Martel-Pelletier et al., 1985). However, human

osteoarthritic cartilage has been shown to contain both an elevated total neutral and acid

metalloproteinase content (Sapolsky et al., 1973; Sapolsky et al., 1976; Martel-Pelletier

et al., 1985 ; Martel-Pelletier et al., 1985) .

Sachs et al. (1982) and Goldberg et al. (1984) described three types of changes in the

metachromatic staining of human osteoarthritic cartilage. Type A is predominantly

characterised by a lack of pericellular staining, giving rise to a halo-like appearance

around the lacunae. Type B involves diminished staining in the interterritorial areas of

the matrix, while Type C is characterised by a more irregular staining of the matrix.

It has been postulated that metalloproteinases which have optimum effects at acid pH

are primarily involved in intracellular and pericellular digestion (Pelletier et al., 1987).

In contrast, neutral metalloproteinases are more likely to also be involved in the

degradation of territorial and interterritorial matrix proteoglycans (Sapolsky et al., 1976;

Bayliss & Ali, 1978; Martel-Pelletier et al., 1984). However, at least one of the acid

metalloproteinases able to be extracted from human articular cartilage, stil l retains 44%

of its maximal activity at pH 7.5 (Woessner & Selzer, 1984; Azzo & Woessner, 1 986).

12

Chondrocyte necrosis and hypocellularity may be associated with areas of mechanical

damage, and hypertrophy and cluster formation have commonly been described around

the periphery of damaged areas (Mcllwraith & V an Sickle, 1 981; Hurtig, 1 988; Shamis

et al., 1 989).

Equine articular cartilage has a very limited capacity to mount a reparative response to

injury (Mcllwraith & Vachon, 1988). The type and effectiveness of repair are

influenced by the size, thickness and anatomical location of the defect (Riddle, 1 970;

Convery, 1972; Grant, 1975; Vachon et al., 1986; Hurtig, 1 988; Shamis et al., 1989;

Desjardins & Hurtig, 1990). Articular cartilage is avascular and thus can not mount a

classical inflammatory response to injury. There are three mechanisms which can

contribute to its repair (Sullins et al., 1985; Hurtig, 1988; Desjardins & Hurtig, 1990):

(1) Intrinsic repair, which relies upon the limited mitotic capability of

chondrocytes and a short-lived increase in the biosynthetic rate of proteoglycans

and collagen.

(2) Matrix flow, which involves the migration of matrix components from the

perimeter to the centre of a lesion.

(3) Extrinsic healing, which involves cartilaginous metaplasia of granulation tissue

originating from subchondral bone, and can occur only when the lesion

penetrates the calcified zone.

Repair of damaged areas of the collagen network is relatively slow, and generally results

in areas of articular cartilage which are structurally less ordered and functionally inferior

(French et al., 1989; Richardson & Clark, 1990). Proteoglycans are synthesised at a

faster rate but require the structural integrity of the collagen framework if the original

functional capability of the cartilage is to be restored. Reduced proteoglycan

concentrations in tissue cultured cartilage explants have been associated with an

increased rate of proteoglycan synthesis (Handley & Lowther, 1977; Sandy et al., 1 980;

Sah et al., 1 988). However, the specific mechanisms regulating proteoglycan synthesis

remain unknown (Tyler, 1985; Schneiderman et al., 1986; Gray & Gottlieb, 1 983).

13

B. USE OF INTRA-ARTICULAR CORTICOSTEROIDS IN THE HORSE

Since their first reported use in the horse by Wheat (1955), corticosteroids hav� gained

widespread use in the treatment of traumatic arthritis and the management of

degenerative joint disease (McKay & Milne, 1976; Hackett, 1982; Trotter et al., 1991 ) .

Clinically they reduce swelling and pain associated with inflammation of the soft tissues

of the joint, resulting in improved function. They do not directly affect the initiating

causes of inflammation, but non-specifically limit its full manifestation. The ability of

corticosteroids to mask the clinical signs associated with joint damage while not

affecting its cause, has lead to much debate and a variety of international regulations

concerning their use in competition horses. The New Zealand Racing Conference, New

Zealand Harness Racing Conference, and New Zealand Equine Federation, all ban any

corticosteroid administration which results in detectable urine or blood concentrations

on competition days.

1 . Anti-inflammatory mechanism of action

Corticosteroids are thought to exert most of their biological activity through inducing,

or inhibiting, the synthesis of new polypeptides via transcriptional regulation (Raz et al.,

1989; Raz et al., 1990). Steroid molecules enter cells and bind to receptors, resulting

in complexes capable of regulating the transcription of specific genes. Induced mRNA

diffuses out of the nucleus, binds to the ribosomes and gives rise to new polypeptides.

The anti-inflammatory effects of corticosteroids are mediated via four main mechanisms:

(i) inhibition of arachidonic acid release, (ii) inhibition of inducible cycle-oxygenase

(COX-2) synthesis, (iii) inhibition of the synthesis and effects of cytokines, and (iv)

inhibition of the generation of granulation tissue.

Corticosteroids are thought to inhibit the release of arachidonic acid from the lipid

bilayer of cell membranes mainly by stimulating the synthesis and release of lipocortin

(Flower & Blackwell, 1979). Lipocortin, a member of the anexin family of calcium

binding proteins, acts by inhibiting free calcium ions activating membrane bound

phosphol ipase A2 from releasing arachidonic acid (Aarsman et al., 1 987; Davidson et

14

al., 1987). Free arachidonic acid is a primary substrate for both the cycle-oxygenase

and lipoxygenase metabolic pathways, which ultimately produce the pro-inflammatory

prostanoids and leucotrienes. Corticosteroids may also inhibit the synthesis and

expression of phospholipase A2 directly (Nakano et al., 1990). However, doubt exists

as to the clinical significance of these mechanisms of action, as phospholipase A2

mediated release of arachidonic acid is not the only, or possibly even the most

important, source of free arachidonic acid in inflamed tissues (Habenicht et al., 1990;

De Witt, 1991 ).

The recent discovery of an isoenzyme of cycle-oxygenase, variably named cyclo­

oxygenase-2 (COX-2), inducible prostaglandin G/H synthase (PGHS-2) or

glucocorticoid-regulated inflammatory prostaglandin G/H synthase (griPGHS), and

whose production is affected by the corticosteroids, has significantly advanced the

understanding of the anti-inflammatory effects of these drugs (Xie et al. , 1991; Kujubu

et al. , 1991; O'Banion et al., 1991). Concentrations of both PGHS-2 MRNA and

enzyme have been shown to dramatically increase in response to growth factors,

cytokines and oncogene activation. Both basal and induced concentrations of PGHS-2

MRNA can be suppressed by dexamethasone, and some evidence exists for both a

transcriptional and post-transcriptional effect. In contrast PGHS-1 expression remains

relatively constant.

Cytokines are defined as soluble peptides produced by one cell affecting the activity of

other cell types (Price et al., 1992). Corticosteroids can inhibit both the production and

effects of many cytokines. Potential sources of cytokines in traumatised or inflamed

joints include the chondrocytes themselves, synovial cells, and inflammatory cells which

have migrated into the synovium (Ollivierre et al., 1986). Several cytokines have been

implicated in the pathogenesis of degenerative joint disease (Price et al., 1992).

Examples of cytokines which have catabolic or pro-inflammatory effects include,

interleukin-1 (IL-l ), the tumour necrosis factors (TNF-a, TNF-13), platelet activating

factor (PAF), and plasminogen activator.

15

Produced by neutrophils, plasminogen Activator facilitates the migration of neutrophils

to inflammatory sites by activating fibrinolysis and hydrolysis of proteins (Haynes, \

1 991 ; Price et al., 1 992). Furthermore, it may play an important role in activation of

the metalloproteases involved in cartilage matrix catabolism (Campbell et al., 1 988).

Platelet activating factor (P AF) is produced by platelets, mast cells, monocytes,

neutrophils, eosinophils, several types of renal cells, and vascular endothelium. It

causes bronchoconstriction, vasodilation, and increases vascular permeability. Also,

P AF promotes platelet and leucocyte aggregation, and is chemotactic for eosinophils,

neutrophils, and monocytes. P AF is not stored by cells, but is synthesized, from cell

membrane phospholipids, in response to adverse stimulation. Synthesis involves

reactions catalysed by phospholipase A2 and lyso-PAF acetyltransferase, both of which

require free Ca2+ ions to become active (Campbell, 1 990). Corticosteroids can inhibit

both the synthesis and effects of P AF. Inhibition of P AF synthesis is thought to be the

result of corticosteroid mediated induction of the synthesis and release of the calcium

binding protein lipocortin (Haynes, 1 99 1 ) .

Interleukin-1 (IL-l) is principally produced by monocyte macrophages but is also

produced by most cell types under stress, including synoviocytes and chondrocytes

(Ollivierre et al., 1 986). Raised synovial fluid concentrations of IL- l have been found

in human and equine patients suffering from osteoarthritis (Wood et al. , 1 983 ; Morris

et al., 1 990). Interleukin-1 stimulates the synthesis and activation of a range of

inflammatory mediators and catabolic enzymes from a variety of different cell types,

and inhibits the synthesis of new proteoglycan aggregates and collagen fibrils by

chondrocytes,(Morris et al., 1 990; MacDonald et al., 1 992). However, some debate

exists over the clinical significance of the chondrocyte mediated catabolic effects of IL­

l at clinically relevant concentrations (Hickery et al., 1 990). Interleukin-1 also causes

activation of T lymphocytes, stimulates fibroblast proliferation, and enhances hepatic

synthesis of ' acute phase' proteins. It is chemotactic for leucocytes and causes

neutrophilia by stimulating release of neutrophils into the vasculature (Price et al.,

1 992). Corticosteroids are thought to reduce the synthesis of IL-l by inhibiting

16

transcription of its MRNA. However, they can also inhibit the actions of IL- l through

a variety of other mechanisms (Haynes, 1991; Lane et al., 1992).

Tumour necrosis factor, originally known as cachectin, has a similar range of

biological effects to IL- l . Mainly produced by monocyte macrophages and

lymphocytes, TNF stimulates bone and cartilage resorption through the induction of

prostaglandins and metalloproteinases (Saklavala, 1986; Brunning & Russell, 1989).

The effects of TNF and IL- l are additive but are not mediated by the same receptor

(Bird & Sak.lavala, 1986). TNF also mediates some of the cytotoxic effects of activated

T lymphocytes, and high plasma concentrations of TNF have been implicated in the

pathogenesis of septic shock. Both the synthesis and some of the effects of TNF can

be inhibited by corticosteroids.

Corticosteroids can also inhibit the synthesis and/or effects of the class of cytokines

more commonly known as the growth factors. These include the insulin-like growth

factors, transforming growth factors, platelet derived growth factor, and fibroblast

growth factor (Price et al. , 1992). Insulin-like growth factor-1 can decrease degradation

and promote synthesis of proteoglycan in cartilage exposed to either IL- l or TNF

(McQuillan et al., 1986; Tyler, 1989). The effects of transforming growth factor p on

the activity of chondrocytes are less clear, but it may play a role in the elevated

proteoglycan synthesis and cell proliferation in osteoarthritic cartilage (van der Morales

& Roberts, 1988; van der Kraan et al. , 1992). Platelet derived growth factor, fibroblast

growth factor, and several others are involved in the complex series of events resulting

in fibroblast proliferation and the generation of granulation tissue (Shoemaker et al.,

1992). The ratio of anticatabolic to antianabolic effects in different clinical situations

is the subject of much debate and is one of the major points of contention concerning

their use.

The exact mechanism of action by which steroids inhibit cell division and even cause

cell death, is not known (Chunekamrai et al., 1989). However, inhibition of normal

metabolism is likely to be an important mechanism of action (Glade et al., 1983). High

concentrations of steroids have been associated with disruption ·of the normal structure

1 7 o f intracellular organelles (Behrens et al., 1 975; Ohira & Ishikawa, 1 986). Cytotoxic

effects have been shown to be both dose and time dependant (Hainque et al., 1 987). \

Certain cell types, for instance lymphocytes, have an inherent increased sensitivity to

these effects (To bin, 1 979; Haynes, 1 991 ) .

Most of the research into the mechanisms of action of corticosteroids has been done in

species other than the horse. However, two equine reports have specifically investigated

what effects clinically recommended doses of betamethasone have on the PGE2-like

activity and leucocyte numbers of carageenan induced acute inflammatory exudate.

Single betamethasone doses (80 f.Lg kg· t , IV), administered several hours prior to the

carageen, had no significant effect on the PGE2-like activity of the subsequently

produced exudate. Furthermore leucocyte numbers in the exudate were unexpectedly

and paradoxically increased in the exudate, both with single dosing and with multiple

dosing for four days before inducing the localised inflammatory reaction. PGE2-like

activity was reduced only after the horses had received either multiple 80 f.Lg kg·1 doses

or a very large single dose ( I mg kg- 1) . The authors concluded that the data failed to

support the hypothesis that the actions of betamethasone in the horse are wholly

attributable to the inhibition of phospholipase A2 and a consequent suppression of

eicosanoid production.

2. Manufacturer recommendations

There are a number of soluble and depot corticosteroid formulations registered in New

Zealand for intra-articular administration. Recommended dose ranges are wide and are

predominantly based on the volume of synovial fluid in the joint being treated. Both

methylprednisolone acetate products available recommend intra-articular doses of 40-240

mg. Neither product specifically stipulates whether this dose is a per joint or a per

horse dose, although the product sheet of Depo-Medrol® suggests that 1 20 mg is the

average dose for a large joint.

3. Pharmacokinetics

\

18

Intra-articular (1/A) injections of methylprednisolone acetate (MPA) are essentially

placed at or close to their site of action. The rapidity, intensity, and duration of the

effects are thus primarily affected by the rates of (a) disintegration of the particles, (b)

dissolution of MPA, and (c) hydrolysis of MP A to methylprednisolone (MP), the active

form of MP A, and not by systemic distribution, metabolism, and elimination

characteristics.

Post-mortem inspections of joint capsules have revealed visible MPA deposits for at

least three days post-injection (Autefage et al. , 1 986), and up to 1 3 days following a

single 1/A administration (Pool et al., 1 98 1 ) . Chunekamrai et al. ( 1 989) reported that

after five weekly I! A injections, crystals of MPA were seen in most of the synovial fluid

aspirates immediately prior to the subsequent weekly administrations. These

observations contrast sharply with the often quoted reports of Wilson et al. ( 1 953) and

Zacco et al. ( 1 954) which concluded that within 2 hours of 11 A administration, most of

the hydrocortisone acetate dose was contained within the synoviocytes or cells in the

synovial fluid, and any remaining drug was almost totally hydrolysed.

Doses of intra-articular depot corticosteroids have traditionally reflected arbitrary and

empirical volume based dosage schedules. The aim of these has been the maximal

suppression of pain and inflammation within a joint for as long as possible, with dosage

intervals determined primarily by pharmacological effect (Autefage et al., 1 986; Trotter

et al., 1 99 1 ) . Only one study has reported the equine synovial fluid kinetics of MP A

and MP following 11 A administration of a MP A formulation (Autefage et al., 1 986).

A total dose of 1 1 1 .2 mg of MP A 1, corresponding to 1 00 mg of MP, was injected into

the right tibiotarsal joint of five adult horses. Even though MP A is practically insoluble

in water, high synovial fluid concentrations (289 ± 284 J.Lg ml" 1) of MP A were recorded

at the first sampling time (2 hours post-injection), and MP concentrations were maximal

(58 .9-379.5 J.Lg ml- 1) at either the first or second sampling time (2- 1 0 hours after

1 Depo-Medrol, 40 mg ml" 1 , Upjohn.

19 administration) . These results indicated that MP A is relatively rapidly hydrolysed to its

active form, presumably by esterases present in the synovial fluid, and should be

considered a rapidly acting drug wh�n administered by the 11 A route in the horse

(Autefage et al., 1 986). The disposition kinetics of MP A were best described by a

biexponential equation of the form: Synovial concentration = Ae·at + Be·Pt, with the

half-time of the second phase estimated to be 1 1 .8 1 ± 7.37 hours.

Even though MPA was present in detectable concentrations ( 1 0-20 ng mL·1 of synovial

fluid) for only 3-6 days in this study, MP was present in pharmacologically significant

concentrations (37 ng mL·1 or greater) from 4.8 to more than 39 days. The disposition

kinetics of MP were also best described by a biexponential equation. After reaching

maximal values at either the first or second sampling time, synovial fluid concentrations

of MP fell progressively for the first five days with a half-time of 9.95 hours, then

decreased more slowly with an apparent half-time of 1 1 5 hours (Autefage et al. , 1 986).

However, very large differences were observed between horses with the half-time of the

second phase varying from 3 7-208 hours; the authors concluded that these differences

were possibly due to variable disintegration of the deposits of MPA in different

locations, and that this might explain the drugs variably sustained action in different

joints. They further concluded that the duration of detectable concentrations of MP ( 1 0

to 20 ng mL- 1 ), following 1/A administration of MPA in a horse, was largely

unpredictable. Thus no pharmacokinetically based recommendations concerning the

inter-dose interval could be offered.

MPA was not detected in plasma (sensitivity 2-3 ng mL- 1 ) at any time after 1/A

administration (Autefage et al., 1 986). However, low concentrations of MP (less than

5 ng mL-1 ) were detected in all horses for about 24 hours following 1/A administration

of MP A. The disposition kinetics of intravenously administered MP have best been

described by a triexponential equation of the form: Plasma concentration = Pe·111 + Ae·at

+ Be·Pt (Autefage et al., 1 986). The half-life calculated from the slope of the terminal

portion of this curve was 1 72 ± 88 minutes (range of values 93-308.5 minutes).

20

Systemic distribution, metabolism, and elimination characteristics were thus considered

not to greatly affect synovial fluid concentrations of either MPA or MP in the horse

following 11 A administration of MP A.

Although MP A is predominantly administered 11 A to ameliorate the effects of joint pain

and inflammation, the effect of joint inflammation on the synovial fluid kinetics of 11 A

administered MP A in horses has not been studied.

4. Clinical use

Corticosteroids can be beneficial in the relief of pain and the treatment or management

of a wide range of intra- and peri-articular conditions. Use of the intra-articular route

of administration allows high concentrations of corticosteroid to be achieved locally in

the synovium and joint capsule, while high plasma concentrations and their consequent

systemic effects are avoided (Autefage et al., 1 986).

Therapeutically, methylprednisolone acetate can be used to inhibit both the exudative

and cellular manifestations of acute inflammation. It can limit the potentially damaging

effects of the inflammatory process on joint tissues, while a more specific therapy

addresses the primary cause of the inflammation (May et al. , 1 987). Through inhibiting

the exudative and the cellular responses to injury, corticosteroids help reduce chronic

joint thickening and scar formation and maintain an effective circulation and nutrient

supply to articular cartilage and surrounding tissues (Mcllwraith & Vachon, 1 988) .

Intra-articular corticosteroids may break the seemingly self-perpetuating cycle of

inflammation initiated by some joint injuries. The permanence of this effect after the

drug has been eliminated, and the effect of corticosteroids on the progression of chronic

inflammation, are less clear (May et al., 1 987). Owen ( 1 980) concluded that IIA

administration of depot corticosteroids may be beneficial if lesions are confined to the

soft tissues of the joint, are not associated with joint laxity, and if the joint is rested for

an appropriate period following therapy.

21

The analgesic effects of I/ A corticosteroids arise as a result of their anti-inflammatory

actions (Higgins & Lees, 1 984). Corticosteroids inhibit the production of several

inflammatory mediators �apable of stimulating or hypersensitizing peripheral nerve

endings. Also, reduced inflammation relieves pressure on capsular insertions (May et al., 1 987). The combined anti-inflammatory and associated analgesic actions can result

in the return of function to an inflamed joint irrespective of its initiating cause.

Consequently, intra-articular depot corticosteroids can be used specifically to mask the

clinical signs of joint inflammation, so as to facilitate the continued training or

competing of an otherwise unsound horse (McKay & Milne, 1 976; Hackett, 1 982).

5. Adverse effects on joints

Despite well documented cases of intra-articular corticosteroids being implicated in the

pathogenesis of progressive joint degeneration, no pathognomonic lesion for so-called

'steroid induced arthropathy' has been described in the horse (Pool et al., 1 98 1 ;

Mcllwraith, 1 989). There is general agreement that most of the steroid arthropathies

result from the intra-articular injection of corticosteroids into joints that either already

have significant cartilage or bone disease, or are not properly rested during and after

therapy (McKay & Milne, 1 976; Pool et al., 1 98 1 ; Mcllwraith & Vachon, 1 990).

Use of corticosteroids in structurally damaged joints, to facilitate continued training and

competition, exposes the compromised joint to a greater risk of further damage (Pool

et al., 1 98 1 ; Trotter et al., 1 991 ). Corticosteroids inhibit tissue repair and normal

turnover of fibrous tissue (Shoemaker et al., 1 992). Intra-articular administration of

depot corticosteroids when chip fractures are present can compromise healing of the

fracture and lead to more extensive and severe joint degeneration, especially if

combined with continued training (Meagher, 1 970). Pre- or post-surgical use can

interfere with normal wound healing and may predispose to post-surgical infection

(Meagher, 1 970). Intra-articular administration of depot corticosteroids in cases of

infectious arthritis compromises the defensive response at the site of infection, and can

ultimately lead to increased agent propagation, dissemination and joint destruction

22

(Tulamo et al., 1 989). Low doses of corticosteroids given over several months interfere

with normal development of joints in foals (Glade et al., 1 983). \

Post inj ection 'flare' is a rare but well recognised possible sequel to intra-articular depot

corticosteroid administration (Owen, 1 980; Hackett, 1 982), as are cases of laminitis

(Vemimb et al., 1 977; Mcllwraith, 1 987). However, the doses normally administered,

and the rate of systemic absorption, are such that systemic immune responses and

adrenal responsiveness are not usually affected (Autefage et al., 1 986).

Intra-articular use of corticosteroids remains controversial, but widespread, in the racing

industry. There is little reliable information on the risk/benefit ratio associated with

their use (Pool et al., 1 98 1 ). However, the following recommendations have been

proposed for the use of intra-articular depot corticosteroid therapy in the horse (Gabel,

1 978; Mcllwraith, 1 987):

( 1 ) They may be indicated for the treatment of synovitis and capsulitis and are very

effective in this regard.

(2) Aseptic technique is mandatory.

(3) Steroids are contraindicated if the joint 1s not radiographed for structural

damage, or if there is instability in the joint.

( 4) The response is less satisfactory where degenerative joint disease is present.

(5) Surgery should be approached cautiously where there is a history of prior

corticosteroid administration in the horse (this is of less serious concern when

surgery is performed arthroscopically)

(6) It should always be remembered that corticosteroids tend to slow the rate of

healing of articular cartilage, joint capsule, l igaments, and bone, and so

additional forced exercise may compound the injuries and damage that have

already occurred

C. EFFECTS OF CORTICOSTEROIDS ON ARTICULAR CARTILAGE

'

23

A variety of species, treatment regimes, and analytical techniques have been used to

investigate the effects of corticosteroids on articular cartilage. Despite much research,

debate, and widespread use in equine practice, there is little objective data on their

potential effects on equine articular cartilage at the concentrations used clinically

(Trotter et al., 199 1 ).

The response of articular cartilage to experimental conditions differs from species to

species. For instance, the half-life of the proteoglycans in rabbit stifle articular cartilage

has been estimated to be as short as 1 0 days (Mankin & Lippiello, 1 969), while in man

the half-life of proteoglycans is estimated to be 300-800 days, depending on anatomical

site (Maroudas, 1 975). There is also wide variation in the magnitude and type of forces

imposed on the articular cartilages of d ifferent joints and different species.

Corticosteroids have a variety of metabolic effects on different tissues and many of

these vary with both the type of corticosteroid and its concentration. Thus, for a given

research result, it is important to recognise the joint type, species, corticosteroid

involved, dose rate, and experimental conditions prevailing. However, information can

still be gleaned from the variety of animal models and techniques used to gain an

insight into what is likely to occur in equine joints.

1 . Normal articular cartilage

One of the primary rationales for the therapeutic use of 11 A corticosteroids is to protect

articular cartilage from damage caused by the products of inflammation. Consequently

it is important to know what effects corticosteroids may have on normal articular

cartilage, and to relate these effects to the size, number, and frequency of

administration.

(i) Chondrocyte morphology

24

Repeated 1/ A administration of large doses of corticosteroid into the stifle joints of

rabbits have resulted in an increased incidence of histologically observable degenerative

changes in treated joints when compared to the 'untreated' contralateral joints (Mankin

& Conger, 1 966; Salter et al., 1 967; Kopta & Blosser, 1 969; Behrens et al., 1 975; Ohira

& Ishikawa, 1 986). Degenerative changes included chondronecrosis, intra-cartilaginous

cysts, fibrillation and fissure development. Ultrastructurally, atrophy of the golgi

apparatus, disruption of the endoplasmic reticulum, and deposition of hydroxyapatite

have also been observed (Behrens et al., 1 975; Ohira et al., 1 986).

Several studies have investigated histological changes in equine carpal articular cartilage

following repeated 11 A administration of a methylprednisolone acetate formulation2 into

clinically normal joints. Two studies reported degenerative cellular changes including

empty lacunae, hypocellularity and intra-cartilaginous cysts (Chunekamrai et al., 1 989;

Shoemaker et al. , 1 992), and another reported 'early osteoarthritic lesions' but no cyst­

l ike degeneration (Marcoux, 1 977). In contrast, Trotter et al. ( 1 99 1 ) found no increase

in the incidence of degenerative lesions in any of the methylprednisolone treated joints.

Repeated systemic corticosteroid administration in foals has also resulted in degenerative

cellular changes (Glade et al., 1 983). Generally, the experiments resulting in a higher

incidence of degenerative chondrocytes involved frequent 1/A administrations of high

doses of corticosteroids (Chunekamrai et al., 1 989; Shoemaker et al., 1 992). Lower

doses of methylprednisolone acetate and/or longer dose interval showed fewer or no

changes in the chondrocytes (Marcoux, 1 977; Trotter et al. , 1 99 1 ).

(ii) Matrix biosynthesis and depletion; in vivo results

Mankin & Conger ( 1 966), using radio labelled glycine, concluded that slightly soluble

hydrocortisone acetate injected intra-articularly profoundly reduces the rate of protein

synthesis in rabbit stifle articular cartilage. The duration of this effect was clearly dose

2 Depo-Medrol, 40 mg ml·1 methylprednisolone acetate, Upjohn Inter-American Corp.

25

dependant. When 0.25 mg was administered the metabolic rate recovered in only 3

days and the opposite joint was not identifiably affected. When 2.5 mg was used \

recovery of protein synthesis took an average of 1 4 days and the opposite joint was also

initially affected. Behrens et al. ( 1 975) investigated the effects of 2-1 2 weekly 1/A

administrations of 25 mg hydrocortisone acetate per knee on rabbit stifle articular

cartilage. Average proteoglycan content, as shown by hexosamine assay, diminished as

the number of injections increased. Also, differences in radiolabel incorporation

between treated and untreated joints demonstrated there were persistent and highly

significant reductions in protein, collagen, and proteoglycan synthesis. Mankin et al.

( 1 972) reported similar effects in rabbit stifle articular cartilage following daily

intramuscular administration of 4.5 mg kg-1 cortisone acetate for up to 9 weeks.

To date, only two studies have compared the biosynthetic rates of cartilage from normal

and methylprednisolone treated equine joints (Chunekamrai et al., 1 989; Fubini er al. ,

1 993). For both investigations, articular cartilage was harvested from a small number

of horses and cultured in media supplemented with I 0 JLCi of Na/5S04 ml· 1 • Counts

per minute (cpm) J.l.g- 1 glycosaminoglycan, and cpm mg·1 (wet weight) of cartilage were

used as relative measures of the rate of proteoglycan synthesis occurring within the

explants. Fubini et al. ( 1 993) reported no significant difference in the rate of

proteoglycan synthesis between explants from control joints versus those from joints

which had had a single 11 A administration of a methylprednisolone acetate formulation

3 weeks previously. In contrast, Chunekamrai et al. ( 1 989) reported the rate of

proteoglycan synthesis of cartilage explants from treated joints was reduced to 1 7 .04%

of control values 1 week after 8 weekly 1 20 mg joinr 1 11 A administrations of

methylprednisolone acetate. Three weeks later the rate of synthesis had increased to

55.3 1 % of control values, and by 8 weeks had further recovered to be 7 1 .28% of

control values. A concurrent experiment using a different joint of the same horses

found that proteoglycan synthesis 1 6 weeks after a single 11 A methylprednisolone acetate

administration was increased, but the difference was not statistically significant

( 1 70.48% of the controls). The horses used in the experiments conducted by

Chunekamrai et al. ( 1 989) were subjected to many large doses of a methylprednisolone

acetate formulation into multiple joints at short intervals. It was likely that

26

methylprednisolone acetate accumulated in the joints which had multiple

administrations. It was also possible that sufficient methylprednisolone was absorbed

to have exerted some systemically mediated effects.

Several studies have histochemically compared cartilage proteoglycan content from

methylprednisolone acetate treated and untreated equine joints. Four of these studies

reported a marked diminution of Safranin-0 staining intensity in the articular cartilage

of the methylprednisolone acetate treated joint (Pool et al., 1 98 1 ; Chunekamrai et al.,

1 989; Trotter et al., 1 99 1 ; Shoemaker et al., 1 992). In contrast Marcoux ( 1 977) could

identify no loss of proteoglycan content in treated joints.

Only Chunekamrai et al. ( 1 989) and Trotter et al. ( 1 99 1 ) have attempted to

quantitatively assay the cartilage proteoglycan content of treated versus untreated joints.

Chunekamrai et al. ( 1 989) used the dimethylmethylene blue assay of Famdale et al.

( 1 982) as a relative measure of the sulphated glycosaminoglycan component of the

cartilage proteoglycan content. One week after eight weekly administrations of 1 20 mg

methylprednisolone acetate per joint, the relative proteoglycan content (J.Lg mg- ' of dry

weight) was about half that of the untreated joints. Trotter et al. ( 1 99 1 ) used Bitter &

Muir's ( 1 962) uronic acid assay as a measure of the chondroitin sulphate component of

the cartilage proteoglycan content and reported that the mean articular cartilage uronic

acid content (J.Lg mg- ' of dry weight) of the methylprednisolone acetate injected joints

was only about 1 7% of that of the lactated Ringer-injected joints.

(iii) Matrix biosynthesis and depletion; in vitro results

Jacoby ( 1 976) cultured human articular cartilage explants from several joints for 8 days

in two concentrations of hydrocortisone succinate (0.0 1 and 0. 1 J.Lg mL- 1) and

hydrocortisone acetate ( 1 000 J.Lg mL-'), and found no difference between treatment

groups and controls in the amount of proteoglycan shed into the media. He concluded

that hydrocortisone does not have a catabolic effect on the proteoglycan content of

tissue cultured human articular cartilage. Proteoglycan content was biochemically

estimated using assays for both uronic acid (Bitter & Muir, 1 962) and hexosamine

27 content (gas liquid chromatography method). Tyler et al. ( 1 982) found that the usual

loss of proteoglycans associated with the culture of porcine cartilage explants was

partially suppressed by the addition of 1 .0 J.Lg mL·1 hydrocortisone succinate to the

culture medium. Tyler et al. ( 1 982) also showed that in the presence of 1 .0 J.Lg

hydrocortisone acetate mL·1 , the explants had less outgrowth of tissue from the cut

surfaces than the controls.

Pool et al. ( 1 98 1 ) cultured explants of equine distal femoral articular cartilage. There

was a marked loss of Safranin-0 staining after 3 days, and a complete loss after 7 days

from explants cultured in the presence of ' low therapeutic concentrations of

methylprednisolone', as compared with both the cultured and uncultured controls.

However, this experiment was reported as part of a more general report and did not

include many details.

2. Articular cartilage of arthritic joints

High concentrations of glycosaminoglycans have been found in the synovial fluid of

horses with osteochondritis, traumatic arthritis and osteoarthritis (AI wan et al., 1 99 1 ) .

Increased concentrations of proteoglycans have also been found in the synovial fluid of

human patients with acute inflammatory arthropathies (Saxne, et al., 1 987; Ratcliffe et al., I 988). Locally injected corticosteroids induced a reduction in the proteoglycan

content of the synovial fluid of human patients with a variety of osteoarthritic conditions

(Saxne et al., 1 986). However, this study did not differentiate whether the reduction

in synovial fluid proteoglycan content was due to a reduced synthesis or decreased

catabol ism.

Total neutral metalloproteinase activity in cartilage from rheumatoid arthritis patients

was roughly eight times that of control subjects (Martel-Pelletier et al., 1 985).

Concentrations of the active forms of the neutral metalloproteinases were also higher

in rheumatoid arthritic cartilage than in normal cartilage, but were not statistically

different. However, when the rheumatoid arthritis patients were split into treatment

groups, those treated with corticosteroids had significantly lower concentrations of the

28 active forms of the enzymes than those not receiving corticosteroids. Furthermore,

concentrations of the active forms of the enzymes present in the corticosteroid treated

group were not significantly different from those of the normal cartilage controls. The

authors concluded that their data strongly supported the theory that neutral

metalloproteinase be involved in the destruction of rheumatoid arthritic cartilage and

indicated corticosteroids can suppress the synthesis and/or activation of neutral

metalloproteinases (Martel-Pelletier et al., 1 985).

In patients with primary osteoarthritis the total acid metalloproteinase activity of

cartilage adjacent to osteoarthritic lesions was greater than cartilage from age matched

clinically normal controls (Pelletier et al., 1 987). The active form of this enzyme was

also elevated at these sites, but the difference was not statistically significant. Patients

treated with corticosteroids had much lower enzyme concentrations than those on other

treatments, but concentrations were not significantly different from those in normal

cartilage. The authors concluded that corticosteroids may regulate the production and/or

activation of all metalloproteinases in cartilage. It was not known whether the effects

of corticosteroids were mediated by a suppression of substances from the synovium or

through a direct effect on the chondrocytes (Pelletier et al. , 1 987).

Equine chondrocytes subjected to adverse culture conditions produce increased

concentrations of the neutral metalloproteinase stromelysin (May et al., 1 99 1 ), and joints

with osteoarthritis have higher synovial fluid glycosaminoglycan concentrations than do

clinically normal joints (Alwan et al., 1 991 ). However, reports are lacking on the

specific effects of corticosteroids on osteoarthritic equine cartilage.

3. Articular cartilage repair

Lesions which penetrate the calcified zone of articular cartilage heal predominantly by

cartilaginous metaplasia of granulation tissue originating from the mesenchymal cells

of the subchondral bone. Corticosteroids have a suppressive effect on both the

proliferation and differentiation of granulation tissue responsible for this extrinsic form

of healing (Meagher, 1 970; Shoemaker et al., 1 992). Articular cartilage lesions which

29 do not breach the junction between the non-mineralised and mineralised cartilage heal

only by intrinsic and matrix flow mechanisms. These repair processes have very l imited

potential for repair of substantial structural damage, and may also be inhibited by

corticosteroids (Tyler et al., 1 982; Shamis et al., 1 989; Shoemaker et al., 1 992).

Shoemaker et al. ( 1 992) compared the effects of four weekly 11 A administrations of 1 00

mg methylprednisolone acetate on (a) normal equine articular cartilage and (b) the

healing of surgically induced osteochondral defects. There were no gross abnormalities

in the articular cartilage adjacent to the induced lesions in the methylprednisolone

treated joints. However, there was a higher incidence of chondrocyte morphological

abnormalities in other areas of the treated joints compared with the untreated

contralateral joints.

The pathogenesis of the cartilage degeneration clinically observed in many

methylprednisolone treated joints remains unexplained. Furthermore, little is known of

the possible dose relationship of the effects of methylprednisolone acetate on

chondrocytes, yet in most other tissues the effects of corticosteroids are very dose

dependant (Haynes, 1 99 1 ) .

D. USE OF PHENYLBUTAZONE IN THE HORSE

30

Phenylbutazone is a non-steroidal anti-inflammatory drug (NSAID) which has been very

commonly used in horses for over 30 years. Its anti-inflammatory and analgesic actions

are used in the management of a variety of soft tissue and musculoskeletal inflammatory

conditions (Lees & Higgins, 1 985; Tobin et al., 1 986).

1. Molecular mechanism of action

The clinical effects of phenylbutazone result predominantly from inhibition of the cyclo­

oxygenase pathway. Following insult, this pathway converts

free arachidonic acid into thromboxane (TxA), prostacycline (PGI2), and the

prostaglandins (Vane, 1 976; Lees & Higgins, 1 985). Phenylbutazone may also scavenge

or interfere with the production of free radicals at sites of inflammation (Auer et al.,

1 990). In addition to being potent mediators of inflammation, prostacycline and the

prostaglandins also potentiate several other important pro-inflammatory mediators and

catabolic enzymes (Higgins & Lees, 1 984).

The specific enzyme to which phenylbutazone binds in the cyclo-oxygenase pathway,

and the nature of this interaction, is the subject of some debate (Tobin et al. , 1 986).

Phenylbutazone is commonly reported to act via its ' inhibition of cyclo-oxygenase'

(Vane, 1 976; Lees et al., 1 986; Hardee & Moore, 1 986). However, some ambiguity

exists in the terminology, as 'cyclo-oxygenase' can be used to describe both the general

metabolic pathway and the first enzyme of that pathway. The cyclo-oxygenase enzyme,

variably named prostaglandin GIH synthase (PGHS) and prostaglandin endoperoxide

synthase, actually has two activities: (a) A cyclo-oxygenase activity, which catalyses a

his-oxygenation of arachidonate to yield prostaglandin G2 (PGG2), and (b) a peroxidase

activity, which catalyses a two-electron reduction of the 1 5-hydroperoxyl group ofPGG2

to produce prostaglandin H2 (PGH2) (Smith et al., 1 992).

Some researchers have proposed that phenylbutazone may inactivate other enzymes in

the cyclo-oxygenase pathway. Flower ( 1 974) proposed that phenylbutazone antagonises

the action of endoperoxidase isomerase, thus specifically blocking the formation of

31

prostaglandin E2 (PGE2). Reed et al. ( 1 985) suggested that phenylbutazone may directly

inhibit the action of both PGHS and prostacyclin synthase, with inactivation of the latter

probably the more clinically important.

The recent discovery of an inducible isoenzyme of PGHS, PGHS-2, which has different

NSAID affinities, may shed new light on the subject (Xie et al., 1 992). In the horse,

inhibition of PGI2, PGE2, and TxA by phenylbutazone is firm evidence that at least in

this species the mechanism(s) of action involves inactivation of one of the PGHS

isoenzymes (Higgins et al., 1 984; Hardee & Moore, 1 986; Lees et al. , 1 986).

There are also various opinions on whether inhibition of the cycle-oxygenase pathway

by phenylbutazone is reversible or irreversible (Ku & Wasvery, 1 973 ; Flower, 1 974;

Lees et al., 1 987). The restoration of thromboxane synthesis by equine platelets, which

are anuclear and thus incapable of synthesising new enzymes, suggests that at least in

this tissue phenylbutazone binds reversibly to PGHS (Kopp et al. , 1 985; Hardee &

Moore, 1 986; Lees et al., 1 987).

The actions of NSAIDs can be divided into central and peripheral, central actions being

analgesic and antipyretic, and the peripheral being anti-inflammatory, local analgesic,

anti-endotoxaemic, and antithrombotic. In contrast to the significant central analgesic

effects of some NSAIDs, the pain relief associated with phenylbutazone use is thought

to result predominantly from its local anti-inflammatory effects. Phenylbutazone has

no direct effect on pain perception in normal tissues. Its analgesic effects are mainly

due to its inhibition of the synthesis of inflammatory mediators such as PGE2 and PGI2,

which normally hypersensitize the nerve endings surrounding injured tissue (Moncada

& Vane, 1 979; Kamerling, et al., 1 983).

Prostaglandin production in acute inflammation is thought to be initially regulated by

the rate of release of arachidonic acid from cell membranes by activated phospholipases.

However, as cycle-oxygenase is physiologically inactivated during catalysis (DeWitt,

1 99 1 ), and pharmacologically by a number of drugs (Smith et al., 1 992), both the initial

concentration of cycle-oxygenase and the rate at which it can be resynthesized soon

32

become the prime determinants of the rate at which arachidonic acid is metabolised to

form the prostanoids (DeWitt, 1 99 1 ).

Small differences in the structure of cyclo-oxygenase pathway enzymes may result in

phenylbutazone having different affinities for the enzymes of the cyclo-oxygenase

pathways of different tissues and different species (Lees & Higgins, 1 985; DeWitt,

1 99 1 ; Simmons et al., 1 993). Furthermore, the rate of synthesis of PGHS in different

tissues can vary by several thousand percent (De Witt, 1 99 1 ). Minimum therapeutic

plasma concentrations of phenylbutazone in man have been estimated to be 50- 1 50 #Lg

ml·1 (Aarbakke, 1 978). Much lower plasma concentrations (5- 1 5 #Lg ml- 1) are associated

with clinical efficacy in the horse (Jenny et al., 1979; Gerring et al., 1 98 1 ). However,

plasma concentrations are not a good measure of the duration of clinical effect, which

normally exceeds pharmacokinetically derived predictions (Lees et al., 1 986).

The products of the cyclo-oxygenase pathway have physiological as well as

pathophysiological functions. Many of the adverse effects of phenylbutazone are

thought to be due to its interference in the normal physiological production of the

prostaglandins and prostacycline, both of which are involved in the maintenance of the

microcirculation of tissues (Whittle & Vane, 1 984; Collins & Tyler, 1 985). However,

it was concluded in a recently reported equine toxicological study that phenylbutazone­

induced gastric erosions in horses are primarily due to a direct toxic effect of the drug

on the endothelium of the microvasculature and intestinal mucosa, and that the

development of erosions is not mediated entirely by a reduction in normal physiological

prostaglandin production (Meschter et al., 1990). The results from this study are in

agreement with those of several other investigations in different species (Whittle, 1 98 1 ;

Rainsford & Willis, 1 982). The adverse effects of phenylbutazone are more severe in

situations of compromised circulation and dehydration (Read, 1 983; Gunsen & Soma,

1 983; Behm & Berg, 1 987), or when given at high dose rates for extended periods of

time (Snow et al., 1 98 1 ; MacAllister, 1 983; Traub et al., 1 983; Mackay et al., 1 983).

Phenylbutazone can also inhibit other enzymes, particularly through its ability to

uncouple oxidative phosphorylation, and at relatively high concentrations suppresses

proteoglycan synthesis (Whitehouse & Haslam, 1 962; Whitehouse, 1 964; Bostrom et al.,

3

4

33

1 964; Domenjoz, 1 966). However, it is not known if phenylbutazone inhibits

proteoglycan synthesis, or other metabolic processes, in equine chondrocytes at the

concentrations achieved clinically in synovial fluid.

2. Manufacturer recommendations

Recommendations concerning phenylbutazone dose, dosage interval, and treatment

period vary widely with both product and route of administration. Recommended

intravenous dose rates vary from 2.2-4.4 mg kg·1 day· 1 for up to 5 days3 to 5 .8- 1 1 .6 mg

kg·1 day·1 for 3 to 5 days4 (both doses rates assuming a 450 kg horse). The latter dose

rate applies to phenylbutazone in a phenylbutazone/isopyrin combination product, in

which with isopyrin, in which phenylbutazone is reported to have a longer plasma

elimination half-life than when administered alone (Jenny et al., 1 979).

Oral dose rates usually incorporate an initial loading dose of up to 8.8 mg kg· 1 , given

in two divided doses on the first day, then progressively lower doses. One product has

a recommendation that the treatment should be reassessed after 1 0 days5• Another

product6 recommends that no more than 4.4 mg kg·1 should be administered daily for

a maximum of 7 days. The other products have no recommendations concerning

duration of treatment. The chronic nature of many of the ailments for which

phenylbutazone is used means it is often administered for extended periods.

Butalone, Techvet laboratories limited.

Isoprin, T echvet laboratories limited.

5 Bute (paste), Techvet New Zealand limited.

6 Myoton granules, Pitman-Moore.

3. Pharmacokinetics

34

Phenylbutazone (4-butyl- 1 , 2-diphenyl-3, 5-pyrazolidinedione) is an enolic acid with

lipophilic properties, a pKa of 4.5 and a molecular weight of 308.37 Da. It has low

aqueous solubility, and consequently most injectable preparations are alkaline solutions

of the sodium salt.

(i) Absorption

Intravenously and orally administered formulations are the most commonly used

phenylbutazone preparations in equine medicine. Bioavailability and the rate of

absorption of orally administered phenylbutazone vary widely between horses and are

influenced by the formulation of the drug and the type of ingesta present (Moss, 1 972;

Gerring et al., 1 98 1 ; Rose et al., 1 982; Sullivan et al., 1 982; Snow et al., 1 983; Bogan

et al., 1 984; Maitho et al., 1 985; Soma et al., 1 985 ; Lees et al., 1 986; Lees et al. ,

1 988). Breed and age differences may also exist, and some products have the

recommendation that lower initial dose rates should be administered to ponies and

immature animals (Lees et al., 1 985).

(ii) Distribution

Plasma kinetics

The plasma disposition of phenylbutazone following intravenous injection can be

described by a two-compartment open model. Phenylbutazone is 96-98 .4% protein

bound in equine plasma (Snow, 1 98 1 ; Gerring, 1 98 1 ). Plasma half-life is estimated to

be 4-6 hours, when doses of around 4.4 mg kg'1 are used. However, the plasma half­

life of phenylbutazone is dose dependant at high dose rates (Pipemo et al., 1 968; Lees

et al., 1 985 ; Maitho, 1 986; Lees et al., 1 986; Lees et al., 1 987). Following a single

intravenous administration, the volume of distribution (V d) in the horse is low, with

estimations ranging from 0. 1 4 1 1 kg'1 (Lees et al., 1 987) to 0.25 1 kg-1 (Rose et al. ,

1 982).

•

35

Following multiple daily administrations the volume of distribution at steady state (V dss) is estimated to be 0. 1 52 I kg-1 (Soma et al., 1 983).

Peak plasma concentrations of 50-70 llg ml-1 are achieved following a single intravenous

4.4 mg kg-1 administration of phenylbutazone, while a similar oral dose produces peak

concentrations of the order of 8- 1 2 /lg ml-1 (Lees et al., 1 985). The time to peak

plasma values (�) following oral administration varies greatly. Concomitant access

to feed generally reduces the peak plasma concentrations, and increases t.na,. (Lees et al.,

1 983; Bogan et al. , 1 984; Maitho et al., 1 986). Peak plasma concentrations are higher

when larger doses are administered, and after several days of administration (Gerring

et al., 1 98 1 ; Soma et al., 1 983; Crisman, 1 990). Peak plasma concentrations as high

as 43 . 8 llg ml-1 have been reported following 4 days of orally administered doses of 8 .8

mg kg-1 (Soma e t al., 1 983). Residual plasma concentrations of 0.2 to 2.0 llg ml- 1

persist 24 hours after a single intravenous administration (about 4.4 mg kg- 1) , and rise

to 2- 1 4 /lg ml-1 after several days of the same dose (Soma et al., 1 985).

Tissue fluid kinetics

The maximal phenylbutazone concentrations found in equine synovial fluid, peritoneal

fluid and tissue cage fluid were always less than the maximal plasma concentrations

(Lehmann et al., 1 98 1 ; Lees et al. , 1 987). Concentrations one third to two thirds those

of corresponding plasma concentrations reflect a relatively good penetration into tissue

fluids, as well as a ready reversibility of the plasma protein binding (Lees et al. , 1 987).

However, the high degree of protein binding may explain why lower concentrations of

phenylbutazone are found in synovial fluid, which has a lower protein concentration,

in comparison to serum phenylbutazone concentrations (Lehmann et al., 1 98 1 ). In

acutely inflamed joints, drug concentrations might be expected to be greater than in

normal synovial fluid, due to increased blood flow, increased vascular permeability and

higher extravascular protein concentrations in sites of inflammation (Higgins & Lees,

1 984).

36

However, synovial fluid phenylbutazone concentrations in human patients treated with

phenylbutazone were significantly related to plasma concentration (55-80%), but not

related to plasma or synovial fluid protein concentration (Farr & Willis, 1 977). Patients

with more actively inflamed joints tended to have lower synovial fluid concentrations.

There have been no studies reporting phenylbutazone concentrations in acutely inflamed

equine joints, but concentrations of around 4 p.g mL·1 have been recorded from normal

equine synovial fluid following single intravenously or orally administered 4.4 mg kg·1

doses (Lehmann et al., 1 98 1 ; Lees et al., 1 987). Due to delayed clearance, synovial

fluid concentrations in an inflamed joint would not be expected to drop as quickly as

plasma concentrations.

Inflammatory exudate kinetics

Phenylbutazone concentrations m induced (tissue cage) acute equine inflammatory

exudate approach corresponding plasma concentrations ( 1 2 p.g mL- 1 ) within 5 hours of

intravenous administration . . However, the clearance rate of phenylbutazone from acute

inflammatory exudate is much slower than that from plasma, and higher concentrations

of phenylbutazone could be expected to persist for longer in exudate than in plasma

(Lees et al., 1 986). Penetration into the inflammatory exudate of more chronic lesions

could be expected to be much slower and corresponding peak concentrations lower.

(iii) Metabolism and elimination

Most absorbed phenylbutazone is metabolised before being excreted, due to its lipophilic

nature and the extent to which it is protein bound. Less than 4% of phenylbutazone is

excreted unchanged in the urine, and the pH of equine urine is not thought to

significantly affect plasma half-life (Pipemo et al., 1 968; Smith et al., 1 985). Biliary

excretion and possibly a degree of enterohepatic cycling are thought to occur. After

intravenous and oral administration of phenylbutazone in two horses, faecal elimination

accounted for 3 7% and 40% respectively (Smith et al., 1 985). Phenylbutazone is

metabolised into three main products, of which only oxyphenbutazone is considered to

retain significant anti-inflammatory activity. The rate of hepatic biotransformation, and

37

possibly biliary excretion, at any given dose, are the main factors determining plasma

half-life of phenylbutazone (Tobin et al., 1 986).

(iv) Oxyphenbutazone kinetics

Oxyphenbutazone is the only metabolite of phenylbutazone that remains therapeutically

active. It is less highly protein bound, and has a much higher renal clearance than

phenylbutazone, but despite these factors has a similar plasma half-life (Gerken & Sams,

1988). There may be differences in the plasma protein and/or tissue binding of

oxyphenbutazone at different concentrations. The V d of oxyphenbutazone following a

single intravenously administered dose of I 0 mg kg-1 was 0.35 I kg-1 (Lehmann et al. , 198 1 ) . However, in another study, estimates of the V dss for oxyphenbutazone varied

from 0 .623 I kg·\ for a 1 . 1 mg kg-1 daily dose rate, to 0.355 I kg-1 for a 4. 4 mg kg- 1

daily dose rate (Gerken & Sams, 1988).

Oxyphenbutazone penetrates readily into body fluids, especially into inflammatory

exudate, in which concentrations tend to be higher than plasma (Lees et al. , 1 986). It

is plausible that oxyphenbutazone concentrations may contribute to the therapeutic effect

of phenylbutazone treatment (Lees et al. , 1987; Gerken & Sams, 1 988). However,

following administration of therapeutic doses of phenylbutazone, oxyphenbutazone

concentrations never exceed those of phenylbutazone in any tissue fluid at any time

(Lees et al. , 1 986).

For a number of years it has been recognised that the clinical effects of phenylbutazone

last longer than would be expected from its plasma half-life. The therapeutic efficacy

of phenylbutazone is primarily related to the drug concentration at the site of tissue

injury. Both phenylbutazone and oxyphenbutazone have a delayed clearance from

inflammatory exudate. As a consequence higher concentrations of both drugs persist

for longer in inflammatory exudate than in plasma. The elimination half-life of

phenylbutazone from inflammatory exudate has been estimated at 24 hours, compared

to a plasma elimination half-life of 4.8 hours. It has been concluded that the plasma

half-lives of phenylbutazone and oxyphenbutazone can no longer be regarded as the

38

most important factors in determining the dose interval for phenylbutazone (Lees et al.,

1 985).

4. Clinical use

At clinically achieved concentrations, phenylbutazone is thought to selectively suppress

many of the mediators of acute inflanunation without adversely affecting white blood

cell migration or cellular repair (Higgins et al., 1 984; Lees & Higgins, 1 986). For these

reasons it is commonly administered to control inflammation and pain associated with

surgical and traumatic wounds, and is routinely used following joint surgery

(Mcllwraith, 1 988; Vachon et al., 1 990; Richardson & Clark, 1 990).

Large intravenous doses of phenylbutazone ( 10- 1 5 mg kg·1 6 hour" 1) have been used

clinically to relieve the visceral pain associated with equine colic and to reduce the

development of endotoxic shock. However, flunixin is much more efficacious in

controlling visceral pain and attenuating the effects of endotoxic shock (Moore et al. ,

1 986; Lees et al., 1 987; Sernrad et al., 1 987), and has largely replaced the use of

phenylbutazone in many of these cases (Burrows, 1 98 1 ; Lees et al., 1 985).

Phenylbutazone is still used in the management of soft tissue inflammation associated

with laminitis, inflamed joints, infections, muscular damage, tendonitis, and ligamentous

strains and sprains (Gabel et al., 1 977; Lees et al., 1 985; Tobin et al., 1 986). Soft

tissue inflammation in an inflamed joint can account for more than 99% of the

resistance to movement (Simon, 1 98 1 ; Tobin et al., 1 986). Phenylbutazone results in

inhibition of further swelling and facilitates less painful joint movement.

Phenylbutazone is not thought to affect the progression of chronic inflammation

(Higgins & Lees, 1 984). The rationale for its use in chronic and degenerative

conditions, such as osteoarthritis, is to reduce the clinical signs of associated

concomitant episodes of acute inflammation (May et al., 1 987).

Regulations concerning the use of phenylbutazone have varied from total prohibition of

any detectable levels to unrestricted use. Presently both the New Zealand Racing and

39

Harness Racing Conferences prohibit any use that results in detectable plasma

concentrations at race meetings or trials, while the New Zealand Equine Federation

permits plasma concentrations of phenylbutazone up to 2 JJ.g ml- 1 on competition days.

5. Adverse effects on joints

Where regulations permit its use, phenylbutazone can be used to facilitate the training

and competing of ' sore' horses (Cannon, 1973). Phenylbutazone is often used after

races to prevent horses from 'cooling out sore' (Tobin, 1 981 ; Tobin et al., 1 986).

However, there is much debate concerning the palliative use of anti-inflammatory drugs

to facilitate the continued training and racing of horses. The drug can also mask clinical

signs associated with more serious injuries, which may be exacerbated by the stresses

of further training or racing (Tobin et al., 1986; May et al. , 1 987; Mcllwraith &

Vachon, 1 988). May et al. ( 1 987) further states that 'use of NSAIDs in chronic

lameness is likely to make prognosis worse for all other treatments except arthrodesis' .

E. EFFECT OF PHENYLBUTAZONE ON ARTICULAR CARTILAGE

Phenylbutazone is often used for long durations to suppress inflammation and pain

associated with joint damage. However, little is known on the effect of phenylbutazone

on anabolic and catabolic processes occurring within normal and osteoarthritic articular

cartilage.

The clinical effects of phenylbutazone have been predominantly associated with its

suppression of prostanoid production (May et al., 1 987). In addition to the prostanoids,

other chemical mediators such as leucotrienes, oxygen radicals, and a variety of

cytokines such as interleukin- 1 and tumour necrosis factor are involved in causing

connective tissue damage associated with joint disease (Price et al., 1 992) . Although

phenylbutazone can induce a degree of local analgesia, it is unlikely that it actually

prevents chronic progression of osteoarthritis in the horse (May et al., 1 987).

40

The l iterature is lacking in accounts of the effects of phenylbutazone on equine articular

cartilage, but several studies have examined the effects of phenylbutazone on bovine and

laboratory animal cartilage (Whitehouse & Haslam, 1 962; Whitehouse, 1 964; Bostrom

et al. , 1 964). Phenylbutazone, at a concentration of 1 54 p.g mL· 1 , inhibited proteoglycan

synthesis in cultured explants of bovine costal and tracheal cartilage, as measured by

relative rates of incorporation of 35S, 32P, 14C-labelled glucose, and 14C-labelled acetate

(Whitehouse & Bostrom, 1 962). Reduced proteoglycan synthesis was attributed to

decreased adenosine-5- triphosphate (A TP) synthesis, due to either inhibition of cellular

respiration or uncoupling of oxidative phosphorylation (Whitehouse & Haslam 1 962;

Bostrom et al., 1 964). Pulver et al. ( 1 956), as reported by Domenjoz ( 1 966), found

transamination of a-ketoglutaric acid with alanine to glutamic acid is competitively

inhibited by phenylbutazone. Domenjoz ( 1 966) proposed that because glutamic acid

furnishes the amino groups for the biosynthesis of glucosamine and galactosamine,

inhibition of its synthesis by anti-inflammatory drugs may be a limiting factor in the

rate of proteoglycan synthesis.

A number of other NSAIDs can interfere with proteoglycan synthesis (Whitehouse,

1 964; Bostrom et al., 1 964; Palmoski & Brandt, 1 979; Palmoski et al., 1 980; Palmoski

& Brandt, 1 983). However, the significance of these effects at clinically utilised

concentrations and treatment durations remain contentious.

F. SUMMARY

41

Methylprednisolone acetate and phenylbutazone are the most common steroid and non­

steroidal anti-inflammatory drugs used for treatment of joint injury in equine athletes.

Their soft tissue mediated clinical effects are well recognised (Higgins & Lees , 1 984 ) .

Whether or not they also confer some degree of chondroprotection is actively debated

(Tobin et al., 1 986; Burkhardt & Ghosh , 1 987; Mcllwraith , 1989). Furthermore , there

is some evidence to suggest their use may actually potentiate the progression of joint

deterioration (Whitehouse & Bostrum , 1 962; Tobin et al., 1 986; Chunekamrai et al. ,

1 989; Trotter et al. , 199 1 ; Shoemaker et al., 1 992). Both the types and mechanisms of

their effects on articular cartilage are subjects of some conjecture and much controversy

(May et al. , 1987; Mcllwraith & Vachon , 1 988; Saari et al. , 1 992).

Relatively few controlled in vivo trials have sought to investigate the effects of MPA

or PBZ on equine articular cartilage. Interpretation of specific drug effects from these

trials has been hindered by their small sample numbers , the types of investigative

procedures performed, and a range of confounding variables. The in vitro maintenance

of tissue allows for a more controlled environment in which the study of specific

interactions can be isolated from confounding variables (Tyler et al. , 1 982).

Furthermore , in vitro methodology facilitates the use of larger sample numbers , and

more precisely controlled dose/response related investigations.

G. OBJECTIVES

The objectives of this study were;

42

( 1 ) to set up and validate an in vitro tissue culture model of equine carpal

articular cartilage, and

(2) to use this model to investigate the dose related effects of

phenylbutazone, and a methylprednisolone acetate formulation (Depo­

Medrol®) l , on chondrocyte viability, and chondrocyte mediated

degradation and synthesis of matrix proteoglycans.

H. HYPOTHESES

43

The following three hypotheses were tested (with respect to chondrocyte viability, rate

of proteoglycan synthesis, and proteoglycan content of the cultured explants) ;

(1 ) the drug is capable of affecting the parameter,

(2) the effect is apparent at clinically relevant concentrations, and

(3) the effect is greater at higher concentrations.

MATERIALS AND METHODS: SECTION ONE

DEVELOPMENT OF THE MODEL

A. ANALYTICAL TECHNIQUES

1 . Biochemical analyses

44

The Gatt and Berman ( 1 966) modification of the Elson and Morgan ( 1 933) colorimetric

method for the determination of amino sugars was to be used to estimate the

glycosaminoglycan content of a large number of cartilage and media samples . It was

therefore necessary to establish that (a) the assay could be performed relatively simply

and without excessive variation within or between batches, and (b) glucosamine ,

galactosamine, chondroitin sulphate and acid digested cartilage produced similar

absorption spectra.

Experiment 1 .

Materials and Methods

Stock solutions of galactosamine, glucosamine and chondroitin sulphate were prepared

as follows :

(a) Deionised water was added to 0.010 g galactosamine (C605H13N , 1 79 Da) to

give 1 00 mL of a stock solution of 1 00 ,.,.g mL-1 galactosamine .

(b) Deionised water was added to 0.0 1 2 g glucosamine hydrochloride

(C605H13NHC1, 215.5 Da) to give 1 00 mL of a stock solution of 1 00 ,.,.g mL-'

glucosamine.

(c) Deionised water was added to 0.027 g chondroitin-6-sulphate sodium

(C14014H20SNNa, 481 Da) to give 1 00 mL of a stock solution equivalent to 1 00

/lg mL1

galactosamine .

I

45

A series of standard solutions containing 6.25 , 12 .5 , 25 , 37. 5 , and 50 p.g mL·1 of

galactosamine or glucosamine were prepared by serial dilution from each stock solution. '

Each of these standard solutions was sealed and kept at 4 • C between uses.

Three similar sized explants of equine carpal articular cartilage were placed separately

into 5 mL pyrex screw-top vials containing 5 mL of 2N HCI . From each of the

chondroitin sulphate standard solutions 2 mL were transferred into vials containing 1

mL of 6N HCI . Both sets of vials were sealed with teflon lined tops, placed in an oven

at 1 oo · c for 12 hours, then allowed to cool to room temperature . From each of the

galactosamine and glucosamine standard solutions 2 mL were transferred into vials

containing 1 mL of 6N HCl, sealed and placed in a boiling water bath for 30 minutes,

then allowed to cool to room temperature.

The amino sugar content of 0.6 mL amounts from each of the vials were then assayed

in triplicate according to the method of Gatt & Berman ( 1966) , as described below.

1 . Ehrlich's reagent was prepared by dissolving 1 g 4-dimethylaminobenzaldehyde

in 30 mL of 1 : 1 ethanol :concentrated HCl (specific gravity 1 . 1 8) .

2 . From each of the acid digested solutions 0 . 6 mL amounts were transferred into

10 mL pyrex screw-top tissue culture vials, to which 0.4 mL amounts of 2M

Na2C03 were added, followed by 0.5 mL of a 2% solution of double distilled

acetylacetone in 1 .5M Na2C03• Reference blanks were prepared in the same

way using 0.6 mL amounts of 2N HCI .

3. Each vial was gently shaken to remove excess C02, tightly sealed with a teflon

lined screw-cap, and placed in a boiling water bath for 20 minutes.

4. The vials were allowed to cool to room temperature, and 1 mL ethanol, and 0.5

mL Ehrlich's reagent were added.

5. Each vial was shaken vigorously to expel excess C02, and its contents

transferred to a 4.5 mL disposable polystyrene spectrophotometer cuvette1 •

Salmond Smith Biolab N . Z. Ltd.

46

6. The relative absorbance of each sample was then read in a spectrophotometer,

at 524 nm, against one of the reference blanks .

The absorption spectra resulting from the three acid digested cartilage samples and the

glucosamine, galactosamine and chondroitin sulphate standards were plotted for

wavelengths from 440 to 600 nm. The absorbance of each of the standard solutions at

524 nm was then plotted against the amino sugar content contained in the initial 0 . 6

mL.

Results and Discussion

Glucosamine and galactosamine produced different absorption spectra (Figure 2 . 1 ) . The

glucosamine standards consistently peaked at a slightly lower wavelength (522-524 nm

as compared with 526-528 nm) , and their peaks tended to be slightly higher than those

of equivalent amounts of galactosamine. The intermediate wavelength of 524 nm was

chosen as the wavelength which best approximated the maximal absorbances of each.

Several researchers have reported glucosamine having a greater absorbance at 530 nm

than galactosamine (Balazs et a/ 1965 , Gatt & Berrnan 1966, Blumenkrantz & Asboe­

Hansen 1976) . The magnitude of this difference varied between reports, and especially

with slight variations of method . In addition, Gatt & Berrnan ( 1966) reported that the

absorption spectrum resulting from glucosamine was of a different shape to that of

galactosamine . However, none of these investigations reported that the peak absorbance

of glucosamine occurs at a slightly lower wavelength than galactosamine. Furthermore,

most researchers have used 530 nm as the 'peak' wavelength for the assay of either

individual or combinations of amino sugars.

There was no difference in the shape of the absorption spectra between the

galactosamine standards and the galactosamine released from the oven-digested

chondroitin sulphate standards (Figure 2.2).

2 Pye Unicam SP8-400 UV /VIS Spectrophotometer.

_;_•_I . '_l_!.....l.. i_ , __ j ___ L_._I. L...J_! _ _ ,_l_.� I_:_ '__! _ _ _ :.,__ '-;-'-'--- L.!_ .i_ __ I I . I ' · i 1 . I i ' i I ! I i : · I l : l I l I j I I ' : : I . I i i ! . . i i ' ! i I ' I L _ __ - -i I I I ' i I ' 1 1 I ., , i I I ' I ' I I ! ; . I I . . I I ! I : i I ! I I ; I l I ! I

_ --- _l __ __j_ ---L _ _ :J_• __ ,__,__,_L _ _ _ � ___ _ L __ __ c__ ! I : I I I ' ! , ' ! 11 ' 'I ! l I I I i I 1 1 I I . , I -�- -� . :-1 ' ) I f- ' 0 , _ ; ! !_ l _1 l' 1- -I ' I ' ' I I I ' : L I l j ' I I ._ j ! ' I ' I ! I I l )"" ' / I I I ' I I

I : i J.L; �m· -� i l l_uj_�� I L.�� _!__ ' ' I I ' ' I r:-1 I ' IT I ' I ' I j ,/ I I ' I - fl ' , I I I , , ' i f . ' I i I L 11 I I : ' _ , l I I 1 , _ : I I _I I I I l l I ' I !

1 ! l ! I I I I I I l ' I I

1m- , : I ! ! ! I : ' 1 1' ' I ' ' , L � l ! : I 1 _ 1- r ,_-� t1+ �-r-' : 11 -·- :-'TI er+ i - , �-

1 ' 1 I I ' I ' · - 1 1 I I j I I ll , I • I 1 I ,_ I 1 1 I I J 1 j I ' 1 : r , 1 1 ' I I

440 480 520 560 6 0 0 Wavelength ( nm)

Blue = glucosamine

Green = galactosamine

47

Figure 2 . 1 Comparison of the absorption spectra o f glucosamine and

galactosamine (from 5-20 p.g) .

440

Figure 2.2

I I ' . I , I , ' I I

480 520

I ' I I I I I I ! - i i ' : I i I I

-�-� :�-:-�.-(: -l ' I I , . I 1 ,� , 1 1 , , ___ _L_;____ P ink = glucosamine I ! I I

l 1 · Green = galactosamine I ! . [ L ·-- '- L -, I ' I I ' I i ' B lue - chondroitin S04 ' I I I ,-1 !-, ,-1 I

5 6 0 6 0 0 Wavelength (nm)

Comparison of the absorption spectra of equivalent amounts of

glucosamine, galactosamine, and chondroitin sulphate.

48

The absorption spectra resulting from the acid digested cartilage samples (Figure 2.3)

were similar in shape to those of glucosamine and galactosamine but peaked at the \

intermediate wavelength of 524 nm (wavelength bandwidth 2.0 A) . None of the other

components of cartilage appeared to interfere with the assay .

The absorbances at 524 nm of each of the three types of standard solutions were

linearly related to amino sugar amounts between 2 .5-20 llg (Figure 2.4) . The

absorbances of the galactosamine standards were similar to those of the glucosamine

standards. However, the absorbances of the chondroitin sulphate standards, which had

been hydrolysed for 12 hours at 1 oo · c, were lower than those of their theoretically

equivalent galactosamine standards (Figure 2.4, Appendix Table 1 ) .

The difference in absorbance at 524 nm produced by equal quantities of galactosamine

and glucosamine was within acceptable limits for the purposes of the proposed

experiments . The reduced colour yield associated with the assay of the galactosamine

component of the acid digested chondroitin sulphate indicates chondroitin sulphate

standards should be used in deference to galactosamine or glucosamine standards, if

absolute values are to be inferred from cartilage digests. However, for comparative

purposes, the use of non-digested galactosamine or glucosamine standards should be

sufficient to estimate the relative amino sugar concentrations of cartilage explants .

Observations made during the experiments

(a) The presence of any remaining effervescence greatly increased both the

magnitude and variability of the absorbances recorded.

(b) The 2 % acetylacetone solution developed a faint brown discolouration, which

affected the assays after several days, especially if left exposed to light.

(c) The acetylacetone stock solution also discoloured over the course of twelve

months ; redistillation removed the discolouration and its associated effects.

(d) The assay was particularly sensitive to changes in acidity resulting from any

leakage during the 12 hour 1 oo · c acid digest (Figure 2.5) .

440 480 520 560 600 Wavelength (nm)

Figure 2.3 Absorption spectra of digested cartilage.

49

0 . 6

0 . 5

-� 0 . 4 � N 1.0 -

Q) 0 . 3 0 s:: a:l ,Q r... 0 1'1.) ,Q 0 . 2 <

0 . 1 � � • I

50

0 . 0 L--------L--------�------�--------�----0 5 1 0 1 5 2 0

Amino Sugar (ug )

Figure 2.4 Comparison of the standard curves of glucosamine, galactosamine,

and chondroitin sulphate.

o Glucosamine (slope = 0.026, intercept = 0.004, r = 0.999)

� Galactosamine (slope = 0.026, intercept = 0.019, r = 0.999)

• Chondroitin sulphate (slope = 0.02 1 , intercept = 0.0 1 9, r = 0.999)

5 1

Figure 2 . 5 The gradation of colours produced by a range o f glucosamine

amounts (2 .5-20 JLg) after being assayed according to the method of

Gatt & Berman. The cuvette containing the yellow/orange solution

is an example of the sensitivity of the assay to any variation in

acidity .

52

These observations were in agreement with other reports (Balazs et al 1 965 , Johnson

1 97 1 , Blumenkrantz & Asboe-Hansen 1976) . Redistillation of the acetylacetone stock \

solution approximately every four months, and preparation of the 2 % acetylacetone

solution freshly on the days it is to be used, should ensure consistency of results . Vials

with evidence of leakage after the oven digest should not be assayed .

Experiment 2.

Materials and Methods

To determine if the stability of the assay chromophore changed with time , amounts of

each of the glucosamine, galactosamine and chondroitin sulphate standard solutions

were acid digested and assayed as described in Experiment 1 . Absorbances at 524 nrn were read within 30 minutes of the addition of Ehrl ich' s reagent, and then subsequently

reread 2 and 23 hours later. Between readings the samples were covered and kept at

room temperature .

Results and Discussion

The results obtained from the first reading were similar to those obtained in Experiment

1 (Appendix Table 2) . However, after 2 and 23 hours the absorbances had decreased

to 90 % and 66 % respectively of the 30 minute values (Figure 2 .6) . The absorbance

of each sample appeared to decrease in proportion to its initial value rather than by an

absolute amount. These results indicate that the time between addition of the last

reagent and the reading of the relative absorbance could be important, especially if

groups of samples are read at different times. Intergroup variation due solely to

chromophore deterioration could be minimised by processing only that number of

samples which could be read in less than an hour, and by ensuring examples of each

group are read at the same time.

0 . 6 53

0 . 5 •

G) (.) 0 . 4 s:l -· c

..0 0 . 3 .�· ··V

J.< 0 , .... -"' -lP 0 . 2 ..0 .�· · < , .. ··

. � · ·

0 . 1 v· ·

0 . 0 0 5 1 0 1 5 2 0

Galactos amine (J.Lg) 0 . 6

0 . 5 • G) (.) 0 . 4 J:: (lj _.

..0 0 . 3 ,_. 0 . -rt) 0 . 2 ..0

< V- · · •

0 . 1 V

0 . 0

0 5 1 0 1 5 2 0

Gluc osamine (J.Lg) 0 . 6

f 0 . 5

CV 0 0 . 4 J:: o . 3 I - ·

c ..0 J.< - -· 0 lP

0 . 2 • . • ·V ..0 < - - - 1111 - ·

0 . 1 •• • • V- • •

"'

0 . 0

0 5 1 0 1 5 2 0

Chon droitin sulphate ( J.Lg)

Figure 2.6 Variations in the amino sugar assay standard curves with time.

0 after 0.5 hour, • after 2 hours, V after 23 hours

2 . Scintillation counting

54

The latter stages of proteoglycan synthesis involve the addition of sulphate molecules

to the glycosaminoglycan side chains. As proteoglycan synthesis is the major metabolic

process using intracellular sulphate, radiolabelled sulphate incorporation has become a

convenient means of studying sulphated proteoglycan synthesis . However, for the

radioactivity emitted from the cartilage to be a representative measure of the rate of

proteoglycan synthesis, the non-biosynthetically bound 35SO/ contained within each

explant must first be reduced to a minimum.

Materials and Methods

Both carpi of an adult thoroughbred gelding, euthanased two hours previously , were

skinned then carefully disarticulated at the middle carpal joint . Using a custom made

cartilage chisel (Figure 2 . 7) , full depth 4 mm wide strips of articular cartilage were

harvested from the central regions of the exposed carpal bones. These were

immediately placed in chilled (4 " C) Dulbecco's modified Eagle medium (DMEM)3 (pH

7 .4) supplemented with a cell culture antibiotic solution (PSK)4 •

A cartilage punch with a 3 mm diameter (Figure 2 . 8) was used to cut 70 explants from

the strips . Each explant was individually blotted dry, weighed o n a Mettler balance5

(reproducibility 0 .05 mg) , and randomly allocated into one of seven groups until each

contained ten explants. The explants in group seven were frozen and stored , for later

3 Dulbecco's modified Eagle medium, high glucose (4 . 5 mg D-glucose per l itre with glutamine but without sodium pyruvate and supplemented with 3 . 7 g sodium bicarbonate per litre). Gibco Laboratories, Life Technologies Inc . Grand Island, N .Y. , 14072 U . S.A.

4 Crystapen® injection, Glaxo N . Z. Ltd . , to a final concentration of 200 IU ml-1 benzylpenicillin Na . Strepomycinsulfat, Boehringer Mannheim Germany, to a final concentration of 200 J.Lg ml-1 streptomycin sulphate . Kanamycin monosulphate, Sigma Chemical Company U . S .A. , to a final concentration of 1 50 1-Lg ml-1 kanamycin.

5 Mettler AT 460 DeltaRange .

/ I

Figure 2.7 Custom made cartilage chisel

55

56

Figure 2 .8 Cartilage punches

57

use as non-cultured cartilage blanks. Groups two, four and six were twice frozen

( -20 · C) and thawed prior to being cultured, to render the chondrocytes non-viable

(Maroudas & Evans 1974) .

Each explant was cultured in a tissue culture well6 containing 0 . 5 mL of DMEM (pH

7 .4) , supplemented with 1 5 % heat inactivated equine serum (ES)7, and 1 0 JLCi mL·1

Na/5S04 8 • The cultures were maintained at 3T C for 24 hours in a humidified

atmosphere of 5 % C02 and air.

Post-culture wash procedures

The explants from group one (containing live chondrocytes) , group two (containing

dead chondrocytes) , and group seven (containing the non-cultured cartilage blanks) were

each acid digested in 4 mL of 2N HCI for 12 hours at 1oo · c without any wash

procedure .

(a) Two wash procedure

Each explant from group three (containing l ive chondrocytes) and group four

(containing dead chondrocytes) was placed in 1 mL of chilled ( 4 · C) phosphate buffered

saline (pH 7 .4) for one hour, then in 1 mL of chilled non-supplemented D MEM for a

further hour, before being acid d igested .

(b) Three wash procedure

Each explant from group five (containing l ive chondrocytes) and group six (containing

dead chondrocytes) was placed in 1 mL of chilled phosphate buffered saline for 40

6 Costar 48 well cell culture cluster, Cambridge, MA 02 140, U . S . A .

7 Gibco' s heat inactivated equine serum, Life Technologies Ltd . Penrose, Auckland New Zealand.

8 Amersham Australia Pty Ltd .

58

minutes, then in 1 mL of chilled serum supplemented DMEM for 40 minutes, and

finally in 1 mL of chilled non-supplemented DMEM for 40 minutes, before being acid

digested.

Emulsion scintillation mixture

The emulsion scintillation m ixture was prepared by d issolving 4 .0 g of 2 ,5-

d iphenyloxazole (PP0)9, the primary solute, and 0 . 1 g of 1 ,4di-[2,-(5-

phenyloxazolyl)]benzene (POPOP)10, the secondary solute, in 667 mL of toluene1 1 and

333 mL of triton X-10012• The mixture was then left to stand for a minimum of 24

hours to ensure dissolution of both solutes.

Scintillation counting procedure

From each of the acid digested cartilage solutions 2 x 1 mL amounts were transferred

into 20 mL glass scintillation vials containing 10 mL of the emulsion scintillation

mixture. Each vial was tightly capped, thoroughly shaken until a homogenous emulsion

formed, and was then left for an hour to allow suspended air bubbles to settle out.

The relative estimate of the amount of 35S within each vial was made by measuring the

fluorescent emissions (counts per minute) between 0 to 655 MeV in a 3 channel

Beckman LS 7500 scintillation counter . Quenched standards1 3 , and a reference

background blank13 were assessed at the same time .

The entire set of samples were then recounted, to confirm that the stability of the

emulsions had not changed during the counting period and affected the counts per

m inute (cpm) recorded.

9• 10BDH laboratory reagents .

1 1 • 12Scintran�\ BDH chemicals Ltd. Poole England.

13 Beckman Instruments Inc. Fullerton CA 92634 U . S . A .

59

Conversion of cpm to degenerations per minute (dpm)

\

Counter efficiency (CE) values for the quenched standards were calculated by dividing

the cpm measured by the theoretically occurring dpm.

= cpm dpm

These counter efficiency values were plotted against the H numbers of the quenched

standards (Figure 2 . 9 and Appendix Table 3 ) , and the resulting 'quench curve' was then

used to estimate the CE value for each sample from its own H number. The dpm of

the non-cultured cartilage blanks (dpm blank) were calculated by dividing each cpm value

by its estimated CE value .

dpm blank =

The dpm values of the blanks were then averaged to provide an estimate of the

background radiation (dpm background ) . The cpm from each vial were converted into dpm

by dividing them by their calculated counter efficiency values and subtracting the

estimate of the background radiation from this total .

dpm = cpm CE

dpm background

The two dpm (dpm rep ! and dpm rep2) values for each sample were then averaged , divided

by the weight of the explant (wgt) , and multiplied by four (the dilution factor) to give

the dpm mg·' of articular cartilage .

dpm mg-1 = 4(dpm rep! + dpm rcp2) 2wgt

1 . 0

·� 0 . 9 ·� -

r::l u

- •

>-.

~ C) 0 . 8 c 4>

·-C) ·---4> •

$.., 0 . 7 4> ...,.> c � • 0 u 0 . 6

0 . 5 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0

H numb e r

Figure 2.9 Quench curve generated by 14C quenched standards.

60

•

3 5 0 4 0 0

61

The percentage of the radioactivity in unwashed explants not due to biosynthetically

bound 35S was estimated by; \

1 . average dpm mg-1 for group two (dead chondrocytes) x _1_ average dpm mg-1 for group one (live chondrocytes) 1 00

The percentage of the radiolabel not biosynthetically bound and still remaining after two

washes was estimated by ;

2 . average dpm mg-1 for group four (dead chondrocytes) x _1_ average dpm mg-1 for group three (live chondrocytes) 1 00

The percentage of radiolabel not biosynthetically bound and still remaining after three

washes was estimated by ;

3 . average dpm mg-1 for group six (dead chondrocytes) x _1_ average dpm mg-1 for group five (live chondrocytes) 1 00

Results and Discussion

Both the two wash and the three wash technique were effective in reducing the non­

biosynthetically incorporated radiolabel to less than 5 % of the incorporated level

(Figure 2 . 10 and Appendix Table 4). The three wash technique appeared slightly more

effective , but the difference was not statistically significant (a =0 .05) .

Theoretically, each wash potentially represented a 1 :200 dilution of the radio label .

However, radio label concurrently transferred on the surfaces of the explant and forceps

may have reduced this dilution factor. The permeability coefficients of sulphate ion in

post-mortem human articular cartilage have been found to be the same whether cells are

alive or dead; thus the diffusion of sulphate is not via active transport (Maroudas &

62

1 6 0 0 0

1 4 0 0 0 0 Live cartilage b:SJ Dead c artilage

1 2 0 0 0 f-- Error bars = SD

1 0 0 0 0 f--bD s

............ 8 0 0 0 !- I s I 0.. i '0 6 0 0 0 1- ['<<"� l �� �,·�,� 1 4 0 0 0 �� T I I '" "· " " ' ' " I ' ', " J I

2 0 0 0 L �� t<�� J [', ��'-, j _, T 0

0 2 3

N umb e r of wash e s

Figure 2.10 Comparison o f wash procedures on biologically incorporated versus

non-bound 358 content of explants of equine articular cartilage.

63

Evans 1974) . The time taken for radiolabelled sulphate to equilibrate with the pools

of non-labelled sulphate ions is thought to primarily depend on its rate of diffusion

through the cartilage matrix . 35SOt has been calculated to achieve 90% of its final

equilibrium concentration across a 1 mm plug of articular cartilage within 6 minutes

(Maroudas & Evans 1974) . Thus, neither the volume of the wash solutions , nor the

time each explant was exposed to them should have been a l imiting factor in this

experiment. The relatively large counts from some of the washed 'non-viable explants'

may have been the result of some cells still remaining viable despite being frozen and

thawed twice .

The use of a suction pipette to remove the radiolabelled media and wash solutions prior

to removal of each explant should minimise concurrent transfer of excess radiolabel on

the surfaces of the explants or forceps . The three wash technique , which includes a

wash in serum supplemented media, should also further help to reduce the concentration

of protein bound drugs in the explants.

3 . DNA Assay

64

Inter-sample variation was to be reduced by expressing the 35S incorporation (dpm) and

amino sugar content of each explant as a function of both its wet weight and DNA

content. It was therefore necessary to establish that the DNA assay could be performed

relatively simply and without excessive variation within or between batches .

Materials and methods

Pyrex vials (5 mL) were prewashed in nitric acid and rinsed in distilled deionised water

to exclude extraneous sources of DNA. Explants of equine articular cartilage were

placed into vials containing 0.05 mL papain14 and 0.95 mL phosphate buffered saline

(PBS) . Each vial was sealed and incubated at 6o · c for 1 2 hours , then allowed to cool

to room temperature .

Standard solutions containing between 2- 10 1-Lg mL-1 DNA were prepared by serially

diluting a liquid preparation of calf DNN5 with sterile PBS (0.05 M NaP04, 2 M NaCl ,

pH 7 . 4) . The DNA content of the digests and standards were assayed according to the

procedure of Labarca & Paigen ( 1980).

1 . The H33258 reagent solution was made by adding Hoechst 3325816 t o PBS to

a final concentration of 1 11g mL- 1 •

2 . Each cartilage digest and each standard solution was briefly sonicated

immediately prior to being assayed.

3 . Using a small bore gel loading pipette tip, 0.05 m L from each o f the cartilage

digests and standard solutions was removed and added to acrylic fluorometric

cuvettes containing 2 mL of the H33258 reagent solution (in triplicate) . These

14 Stabilised liquid papain, Salmond Smith Biolab Ltd .

15 Calf DNA, Boehringer Mannheim N .z. Ltd .

16 Hoechst 33258 , (2-[2-( 4-hydroxyphenyl)-6-benzimidazolyl-6-(1 -methyl-4-piperazyl )-benzimidazol . 3 H Cl , BrdU, bromodeoxyuridine.

65

were capped and inverted several times, to ensure even mixing , and then

allowed to settle for 30 minutes.

4 . Fluorescence was measured in a fluorometer with the excitation wavelength set

at 356 nrn and the emission wavelength set at 458 nrn .

Results and Discussion

The 0.05 rnL amounts of the DNA standard solutions contained 100, 200, 300, 400,

and 500 ng of DNA . Fluorescence was linearly related to the amount of DNA with

little variation between replicates (Figure 2 . 1 1 & Appendix Table 5) . The DNA content

of the digested cartilage aliquots ranged from 64 . 1 - 166 .7 ng (mean = 1 20 .9 , SD =

23 . 8 ng) . However, there was substantial variation between the replicates of the

digested cartilage samples Table 2 . 1 . This variation could have been caused by either

a lack of homogeneity in the digested cartilage solutions or problems with assay

technique and or equipment . Either way the results indicated further assessment of this

assay would be necessary before it could be included in the final experimental protocol .

Sample Rep 1 Rep 2 Rep 3 Mean DNA

(Relative fluorescence) (ng)

1 0 . 10 0 . 19 0 . 16 0. 15 98.3

2 0.24 0 . 17 0 .25 0.22 146.2

3 0 .20 0 .21 0 . 14 0. 18 1 18.8

4 0 . 13 0 . 18 0 . 18 0. 16 105.2

5 0.20 0 . 13 0 . 16 0. 16 1 05.2

6 0 . 17 0 . 1 1 0 . 19 0.16 105.2

7 0 .20 0 . 15 0 . 19 0.18 1 18.8

8 0 . 1 8 0 . 1 2 0 . 1 8 0. 16 105.2

9 0 . 1 5 0 . 1 8 0 . 1 8 0 .17 1 12.0

10 0 . 19 0 .20 0 . 1 8 0 .19 125.0

Table 2 . 1 DNA assay of digested cartilage samples

66

1 . 0

0 . 8 •

I

CD 0 . 6 I (.)

s:: CD • (.) tf.l CD $-< 0 ;:::!

0 . 4 -r.:.. / 0 . 2

/ 0 . 0

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

DNA (ng )

Figure 2 . 1 1 Standard curve for calf thymus DNA assayed according to the

method of Labarca and Paigen (1980) .

67 B. MEASUREMENT OF PROTEOGLYCANS IN THE CULTURE MEDIA

1 . Factors affecting the amino sugar assay

It was intended to measure the amount of proteoglycan released from each explant into

the medium during culture. It was therefore necessary to establish whether the culture

medium, drugs being tested , or serum interfered with the amino sugar assay .

Experiment 1 .

Materials and Methods

1 . DMEM was added to 0.0120 g glucosamine hydrochloride to a final volume of

100 mL to give a stock solution of 100 J.Lg mL-1 glucosamine in DMEM .

Standard solutions containing 0, 12 .5 , 25 , 37 . 5 , and 50 J.Lg mL-1 glucosamine

were prepared by serially diluting this stock solution with DMEM .

2. From each of these standard solutions 0.4 mL amounts were mixed with 0.2 mL

6N HCl , and then assayed in triplicate for amino sugar content according to the

method of Gatt & Berman (1966) as described earlier.

3. In addition, 1 .5 mL of each standard solution was added to a 5 mL pyrex vial

containing 0.5 mL 6N HCI . These were sealed, placed in a 100 ' C oven for 12

hours , then 0.6 mL amounts were assayed in triplicate for amino sugar content.

Results and Discussion

The absorption spectra from the glucosamine standards dissolved in DMEM (Figure

2 . 13 and Appendix Table 7) shared some similarity to those obtained from glucosamine

standards dissolved in water (Figure 2 . 1 2 and Appendix Table 6) , but were more

variable and contained an extra peak at approximately 556 nm . In contrast, the

absorption spectra from the oven-digested glucosamine standards (Figure 2 . 14) had a

d ifferent shape, were much more variable , and were less representative of their

glucosamine content.

Q) 0 ..... ,.... � ..0 14 0 I'll ..c <

I ! __ L ' __ L __i __ L _

4 4 0

! I I I I . I � I .

4 8 0 5 2 0 5 6 0 Wavelength (nm)

' i : 6 0 0

Blue = 1 5 p.g

P ink - 10 p.g

Green = 5 p.g

Figure 2 . 12 Absorption spectra of glucosamine standards disolved in water.

Q) 0 � � ..c 14 0 I'll ..c <

4 4 0

: ' i I

!

i ! ' 4 8 0

I i I I , I

r 1 - -, - ! !.' !. I I !

� i ' I '

5 2 0

' i I I !

5 6 0 Wavelength (nm)

6 0 0

Black = 20 p.g

Blue = 15 p.g

Pink = lO p.g

Green = 5 p.g

Figure 2 . 13 Absorption spectra o f glucosamine standards disolved in DMEM.

Q) 0 � Blue = 1 5 p.g � ..c 14 Pink l O p.g 0 = I'll ..0

Green = 5 p.g <

4 4 0 4 8 0 5 2 0 5 6 0 6 0 0 Wavelength (nm)

68

Figure 2.14 Absorption spectra of glucosamine standards disolved in DMEM and

assayed after oven digestion in 2N HCI.

69

The absorption spectra from the non-oven-digested standards indicated that at least one

of the components of DMEM resulted in a chromophore which directly interfered with

the assay of glucosamine . In comparison, the absorption spectra from the oven-digested

standards suggested acid digestion of the medium resulted in a product or products

which interfered with the assay of glucosamine. The results were not unexpected

considering the experiment attempted to assay microgram amounts of amino sugar from

a culture medium containing milligram amounts of a variety of sugars and amino acids

(Table 2 .2) .

Experiment 2.

Culture media used for explant culture are usually supplemented with serum . The aim

of this study was to ascertain if equine serum (ES) interfered with the assay of standard

amounts of glucosamine .

Materials and Methods

1 . The stock solution containing glucosamine disolved in DMEM ( 1 00 J.Lg mL1)

was further diluted with DMEM and ES to make standard solutions of 0 , 1 2 . 5 ,

2 5 , 3 7 . 5 , and 5 0 J.Lg mL·1 glucosamine in DMEM + ES .

2. From each of the standard solutions 1 .5 mL were added to 5 mL pyrex vials

containing 0.5 mL 6N HCl. These were sealed and kept at wo · c for 12 hours ,

before 0.6 mL amounts were assayed in triplicate for amino sugar content .

Results and Discussion

The formation of black suspended particles during the oven digestion precluded assay

of the serum supplemented glucosamine standards. These suspended particles , thought

to be partially oxidised serum protein, caused scatter and prevented the reading of a

meaningful absorbance .

70

Table 2.2 Chemical composition of Dulbecco's modified Eagle medium (DMEM).

Components Concentrations (mg 1·1 )

Glucose 4500

Phenol red 1 5

L-Arginine. HCI 84

L-Cystine . HCI 63

L-Glutamine 584

Glycine 30

L-Histidine . HCI.H20 42

L-lsoleucine 1 05

L-Leucine 1 05

L-Lysine.HCI 1 46

L-Methionine 30

L-Phenylalanine 66

L-Serine 42

L-Threonine 95

L-Tryptophan 1 6

L -Tyrosine 2Na.2HP 1 04

L-V aline 94

D-Ca Pantothenate 4

Choline chloride 4

Folic acid 4

i-Inositol 7 .2

Niacinamide 4

Pyridoxal . BC! 4

Riboflavin 0.4

Thiamine.HCI 4

CaCI2 (anhydrous) 200

Fe(N03).9H20 0. 1

KC! 400

MgS04 (anhydrous) 98

NaCI 6400

NaHC03 3700

NaH2P04.H20 125

71

Bovine albumin (500 f.Lg) had previously been reported not to interfere with the

detection of galactosamine from chondroitin sulphate (20 J.Lg) in samples acid digested

for 30 minutes at 120 ' C (Blumenkrantz & Asboe-Hansen 1976) . In contrast to the

experiments performed by Blumenkrantz & Asboe-Hansen ( 1976) , each sample assayed

in this experiment contained approximately 3600 Jlg of equine serum albumin, plus the

other components of serum, and was digested for 12 hours at 1 00 ' C.

Experiment 3 .

I t was also possible that either the actual drugs to be tested , o r a component o f their

formulations, could interfere with the assay . The aim of this experiment was to

determine whether common formulations of the d rugs l ikely to be used on the explant

model directly affected the assay .

Materials and methods

1 . Aqueous solutions were prepared which contained (a) 100 J.Lg mL1

phenylbutazone sodium17 , (b) 1000 f.Lg mL·1 betamethasone sodium phosphate18 ,

(c) 1000 J.Lg mL·1 gentamicin19 and (d) 50 J.Lg mL1 flunixin meglumine20•

2. From each of these solutions 1 . 5 mL were added to 5 mL pyrex vials containing

0 .5 mL 6N HCl , which were sealed and kept at IOO ' C for 12 hours , then 0.6

mL amounts were assayed for amino sugar content, and the resulting absorption

spectra plotted for wavelengths from 440-600 nm .

1 7 Butazone, Phenylbutazone sodium, C-Vet Ltd .

1 8 Betsolan Soluble, Betamethasone sodium diphosphate, Pitman-Moore .

19 Gentamicin 50, Gentamicin sulphate, R.W.R Veterinary Products Ltd.

2° Finadyne , Flunixin Meglumine , Schering Corporation.

72

Results and Discussion

The peak absorbance (524 run) of the gentamicin solution was many times greater than

the peak absorbances of the glucosamine standards previously assayed . The result was

not unexpected considering gentamicin contains amino sugars as part of its molecular

structure . The phenylbutazone formulation resulted in the formation of an orange

precipitate during the assay procedure, precluding the spectrophotometric reading of a

meaningful absorbance. However, the betamethasone and flunixin meglumine solutions

had comparatively small absorbances at 524 run , and the shape of the absorption

spectrum of each was different to that of glucosamine .

2 . Methods t o reduce media and drug interference

The 'results from the previous three experiments indicated that further processing was

needed to remove or reduce media, serum and drug concentrations before the amino

sugar content of proteoglycans released during culture could be assayed .

Experiment 1 .

A number o f the components o f DMEM may have contributed to its interference of the

assay of amino sugar standards. Selective omission of certain media components and

aqueous dilution were thus investigated as means of reducing this interference.

Materials and Methods

1 . A stock solution of chondroitin-6-sulphate in DMEM, equivalent to 500 pg mL-1

galactosamine , was prepared by adding DMEM to 0 . 1 35 g chondroitin-6-

sulphate sodium (C14014H20SNNa, 481 Da) to give a final volume of 1 00 mL.

2 . The chondroitin sulphate stock solution was serially diluted with volumes of

DMEM to produce a series of solutions containing the equivalent of 225 , 375

and 500 p.g mL-1 galactosamine in DMEM. Each solution was then diluted 1 :9

with water to give chondroitin-6-sulphate standards, dissolved in 1 0 % DMEM,

73

which theoretically contained the equivalent of 1 8 . 7 5 , 3 7 . 5 , and 50 J.Lg mL-1

galactosamine.

3. Phenol red was added to 100 mL of deionised water (pH 7 .4) unti l the colour

of the resulting solution approximated that of DMEM (pH 7 .4) .

4. Two further solutions of DMEM were prepared; (a) DMEM supplemented with

L-glutamine (2000 J.Lg mL-1) , and (b) DMEM diluted 1 :4 with water.

5. From each of the chondroitin-6-sulphate standard solutions 1 . 5 mL were added

to 5 mL pyrex vials containing 0 .5 mL 6N HCl , which were sealed and kept at

1 oo · c for 12 hours , then 0 .6 mL amounts of each were assayed for their amino

sugar content .

6. Amounts ( 1 . 5 mL) of the (a) aqueous phenol red solution, (b) DMEM , (c)

DMEM supplemented with L-glutamine , and (d) 1 :4 aqueous dilution of DMEM

were added to 5 mL pyrex vials containing 0 . 5 mL of 6N HCI . These vials

were sealed and placed in a boiling water bath for 30 minutes, then 0 . 6 mL

amounts were assay for amino sugar content according to the method of Gatt &

Bennan ( 1 966) , with the exception that their absorption spectra were plotted

relative to a non-assayed distilled water blank .

Results and Discussion

The use of water blanks instead of assayed blanks resulted in larger more readily

comparable absorbances at 524 nm. DMEM had a similar absorption spectrum to

glucosamine and galactosamine, but had an additional peak at around 564 nm (Figure

2 . 1 5) . There was no difference between the absorption spectra of DMEM and DMEM

supplemented with L-glutamine . The aqueous phenol red solution had a single small

absorption peak at approximately 508 nm , in comparison to the dual peaks at

approximately 524 and 564 nm produced by DMEM (Figure 2 . 15 ) , indicating phenol

red was not the most important component of DMEM interfering with the assay of

amino sugars . The absorption spectra of the oven-digested chondroitin-6-sulphate

standards dissolved in a 1 0 % aqueous solution of DMEM were similar to chondroitin-6-

sulphate standards dissolved in water.

74

I I I , - -Galactosamine equivalent I I I

Blue = 40 p.g

P ink = 30 p.g

Q) Green = 22 . 5 p.g C) Q � Black = 1 5 p.g ,.0 � 0 B lue = 7 . 5 p.g en i ,.0 < i P ink = 3 . 75 p.g ---- j ----I

Green = 1 . 5 p.g i Black = 0 . 0 p.g

4 4 0 4 8 0 5 2 0 5 6 0 6 0 0

Wavelength (nm) )

Figure 2 . 1 5 Absorption spectra of chondroitin sulphate standards disolved in DMEM following dialysis and oven digestion in 2N HCl (2 .0 A) .

4 4 0 4 8 0 5 2 0 5 6 0 6 0 0 Wavelength (nm)

Green = DMEM Pink = gentamicin

Black = H20 b lank

Figure 2 . 1 6 Absorption spectra o f D:MEM and gentamicin following dialysis and

oven digestion in 2N HCl (relative to a water blank, 2 .0 A) .

75

The results indicate little advantage would be gained by excluding either L-glutamine

or phenol red from the DMEM formulation. However, the use of processing techniques

which selectively dilute the culture medium with respect to the proteoglycans could

greatly reduce the interference the culture medium had on this assay .

Experiment 2.

Dialysis was investigated as a means of reducing the interference caused by either

culture media components or supplemented drugs , in the assay of the amino sugar

component of chondroitin sulphate standards .

Materials and methods

1 . Dialysis tubing21 was prepared by boiling i t in 0.05 mol l-1 Na2C03 and 0 .01 mol

l-1 sodium ethylenediaminetetra-acetic acid (EDTA) for 1 0 minutes, followed by

1 0 minutes in boiling deionised water, with a final rinse in cold deionised water.

The tubing was then cut into 5 cm lengths , and one end was folded over and

clamped to form open ended sacks .

2. Portions of the chondroitin sulphate in DMEM stock solution (500 f..Lg mL1

galactosamine in DMEM) were diluted with DMEM to produce a series of

standard solutions containing the equivalent of 0- 100 f..Lg mL1 galactosamine in

DMEM .

3. Portions of the chondroitin sulphate in water stock solution (100 f..Lg mL1

galactosamine) were diluted with water to produce aqueous standard solutions

containing the equivalent of 0- 100 f..Lg mL1 galactosamine .

4. From each of the two sets of standard solutions 1 mL volumes were placed in

dialysis sacks which were sealed and suspended for 24 hours in three changes

of deionised water at 4 • C, each equivalent to 100 times the combined volume

of the samples .

21 Union Carbide 10 mm cellulose dialysis membrane, molecular weight cut off approximately 1 2-20 kDa .

76

5 . Volumes ( 1 mL) of (a) DMEM, (b) water, and (c) a 1000 p.g mL-1 aqueous

solution of gentamicin were transferred into dialysis sacks in triplicate and

individually dialysed for 24 hours against two changes of deionised water ( 4 · C) .

6. The contents of the dialysed sacks were emptied into graduated vials ,

standardised to 1 .2 mL, and 1 mL transferred to 5 mL pyrex vials containing

0 . 5 mL 6N HCI . These vials were sealed and placed in an oven kept at 1 00 " C

for 1 2 hours , then 0 . 6 mL amounts were assayed in duplicate for amino sugar

content, and the absorption spectra plotted for wavelengths from 440-600 run .

Results and Discussion

Chondroitin sulphate was used instead of glucosamine as its molecules were more l ikely

to be retained within the dialysis sacks because of their larger size . The molecular

weight of the chondroitin sulphate used was not known, but the average molecular

weight of chondroitin sulphate molecules extracted from articular cartilage has been

estimated to be around 20 kDa (Stockwel l , 1979; Camey & Osbome, 1 99 1 ) . The

molecular weight cut-off range for the dialysis tubing used was 1 2-20 kDa . Pore sizes

of d ialysis membranes are not uniform, but can be expected to have a normal

distribution around the mean pore size . Boil ing of the dialysis tubing prior to its use

reportedly ensures a more uniform pore size .

Dialysis removed much of the interference caused by DMEM in the assay of the

galactosamine component of the chondroitin sulphate standards (Figures 2 . 16 & 2. 1 8) .

The use of dialysis resulted i n only small losses of chondroitin sulphate but a dramatic

reduction in the concentration of both DMEM components and gentamicin left within

each sack (Figure 2 . 1 7) . However, the need to re standardise the volume of each

dialysed sample coupled with the extra number of sample manipulations associated with

the use of dialysis could be expected to increased the overall experimental error, and

produced more variable results.

4 4 0 4 8 0 5 2 0 5 6 0 Wavelength ( nm)

6 0 0

77

Red = DMEM

Blue(light> = D MEMCdialysed>

Blue(dark) = gentamic�diaJysed)

Figure 2 . 17 Absorption spectra of DMEM and gentamicin following dialysis and

oven digestion in 2N HCl (relative to a H20 blank, A = 2.0)

78

0 . 5

0 . 4 •

• -

Ei � "''t 0 . 3 N ID - •

Cl) t) � • a:l .0 0 . 2 � 0 en .0 Q

<

/ 0 . 1 I Q

I Q

I . . •

0 . 0

0 5 1 0 1 5 2 0 2 5

G e l e c t o s e m i n e ( u g )

Figure 2 . 1 8 Comparison of chondroitin sulphate standards disolved in water and

D:MEM after dialysis and oven digestion.

• Chondroitin sulphate standards in water (solid regression l ine) .

v Chondroitin sulphate standards in DMEM (dashed regression l ine) .

79

3 . Separation of chondroitin sulphate from solutions containing serum proteins

D ialysis could be expected to remove most of the interference in the assay of amino

sugars from DMEM and drugs with relatively small molecular weights . However, the

molecular weights of equine serum proteins are much too large to pass through any

dialysis membrane which would still retain both glycosaminoglycan and proteoglycan

molecules. Other processing techniques were thus needed to separate

glycosaminoglycan and proteoglycan molecules from equine serum proteins .

Experiment 1 .

The use of papain to hydrolyse the serum proteins into smaller peptide fragments was

investigated as a means for reducing the interference equine serum had on the assay of

amino sugars .

Materials and methods

1 . A papain solution (50- 1 50 units mL·1 activity) was prepared by dissolving 500

mg of papain22, 1 00 mg of d isodium EDTA, and 1 0 mg of cysteine

hydrochloride in water to a final volume of 1 0 mL.

2. An aqueous solution of equine serum ( 1 5 % ) was divided into 1 mL aliquots to

which 0. 1 -0 .5 mL amounts of the papain solution were added . Papain blanks

were prepared by adding 0 . 2 mL of the papain solution to 1 mL volumes of

deionised water. Both sets of solutions were incubated at 65 ' C for 1 2 hours.

3 . Following incubation, 1 mL amounts were transferred into 5 mL pyrex vials

containing 0 . 5 mL 6N HCI, which were sealed and kept at 1 00 · C for 1 2 hours,

then 0 . 6 mL amounts were assayed in duplicate for amino sugar content.

22 Crude papain, 1 -3 units mg ·1 activity, S igma Chemical Company U . S . A.

80

Results and Discussion

Treatment with papain did not prevent the formation of a black precipitate in any of the

equine serum supplemented solutions , and increased amounts of papain may be needed

to hydrolyse this amount of protein into small enough fragments to prevent the

formation of a black precipitate. However, the assay of the papain blanks resulted in

a much higher absorbance at 524 run than any of the amino sugar standards previously

assayed (Figure 2 . 1 9) , and the use of greater amounts of papain to further hydrolyse

the serum proteins would be expected to produce even greater interference in the assay

of the amino sugar content of proteoglycans.

The use of papain in combination with the Gatt & Berman ( 1 966) method for assaying

amino sugars has previously been reported (Richardson & Clark 1 990) with no mention

of an interfering chromophore being formed . The papain source used in this

experiment was not highly purified and it is possible that the chromophore resulted from

impurities rather than the papain itself.

Experiment 2.

The use of a more refined papain formulation was investigated to determine whether

the papain or a contaminant was responsible for forming the interfering chromophore .

Materials and methods

1 . An aqueous solution of equine serum ( 1 5 % ) was divided into 1 8 x 1 mL aliquots

to which 0.05-0 .5 mL amounts of a refmed papain solution23 were added , and

the solutions incubated at 65 · C for 1 2 hours . Each solution then had 1 mL

transferred to a 5 mL pyrex vial containing 0.5 mL 6N HCl , which was sealed

and kept at 1 00 · C for 12 hours , before 0.6 mL amounts were assayed in

duplicate for amino sugar content .

23 Stabilised liquid papain, 100 units mt·1 activity , Salmond Smith Biolab N . Z . Ltd

Cl) 0 c 0 ..0 '-0 Cl! ..0 -<

440 4 8 0 5 2 0 5 6 0 6 0 0

Wavelength (nm)

81

6 4 0

Figure 2 . 19 Absorption spectrum produced by a crude papain blank solution.

82

2. From each of the previously prepared glucosamine standards 4 x 0.4 mL

amounts were added to 5 mL pyrex vials containing 0 . 2 mL 6N HCI . To half

of these 0 .05 mL of the papain solution was added , and to the other half 0 .05

mL of water was added . Each vial was sealed , placed in a boiling water bath

for 30 minutes, cooled to room temperature , then assayed for amino sugar.

Results and Discussion

None of the concentrations of papain prevented the formation of a black precipitate in

the equine serum supplemented solutions . The absorption spectrum from this papain

formulation was similar to that produced by the crude papain solution except the

absorption peaks were generally at slightly lower wavelengths (8 to 1 2 nm) , and the

peaks corresponding to the previous peaks at 500 and 568 nm were less pronounced

(Figure 2 . 20) . The absorbance at 524 nm produced by the 0.05 mL amounts of the

papain solution was constant and additive to that produced by the glucosamine standards

(Figure 2 . 2 1 ) indicating this papain concentration could possibly be used in this assay

if the amino sugar concentrations were relatively high in comparison to the amount of

protein to be digested , as would be the case in digests of explants of articular cartilage .

Experiment 3.

The addition of trichloroacetic acid (TCA) is commonly used to precipitate proteins out

of solution. The aim of this experiment was to find the lowest concentration of TCA

capable of precipitating enough of the proteins out of a 1 5 % solution of equine serum

to prevent the formation of the black precipitate during the subsequent acid digest .

Materials and methods

1 . TCA solutions equivalent to 24 , 12 , 6 and 3 % of saturation were prepared and

0. 5 mL amounts of each of these were added to microcentrifuge vials containing

1 mL of a 1 5 % aqueous solution of equine serum. These were kept at 4 • C for

2 hours , then centrifuged at 1 900 g for 5 minutes.

480 5 2 0 5 6 0 Wavelength (nm)

! i . i

i - i j

6 0 0

83

Green = glucosamine + papain

Pink = glucosamine

Figure 2 .20 Comparison of the absorption spectrum of glucosamine, and a

glucosamine + papain combination.

0 . 9

0 . 8

0 . 7

E 0 . 6 c "'<t N I[) 0 . 5

Q) () c 0 ..0 .... 0 If) ..0 <(

0 . 4

0 . 3

0 . 2

0 . 1

84

•

0 . 0 �--�--�----�--�---L--�----�--�--�--� 0 2 4 6 8 1 0 1 2 1 4 1 6

Glu c o s amine (ug )

Figure 2.21 Effect of papain on the assay of glucosamine standards.

t:. Glucosamine + 50 JLI papain

• Glucosamine

1 8 2 0

85

2. Supernatant ( 1 .2 mL) from each vial was transferred to a pyrex vial containing

0 . 6 mL 6N HCl , which was then sealed and kept at t oo · c for 1 2 hours .

Results and Discussion

The 0.5 mL amounts of the 24, 12, 6 and 3 % TCA solutions, when added to the 1 mL

volumes of the 1 5 % serum solution, equated with final TCA concentrations of 8 , 4, 2 ,

and 1 % . All concentrations were effective in causing at least some degree of

precipitation initially, but only the 2, 4 and 8% final concentrations were completely

effective in preventing the formation of a black precipitate during the subsequent 1 2

hour acid digest at 1 00 · c . Use o f TCA to precipitate the equine serum out o f solution

would require further processing of the supernatants to then remove the TCA itself,

before these samples could have their amino sugar content assayed by the method of

Gan & Berman ( 1 966) .

Experiment 4.

The use of TCA precipitation of the serum proteins followed by dialysis of the resulting

supernatants against deionised water was investigated as a means of reducing the

interference from both equine serum (ES) and DMEM on the estimation of the amino

sugar content of chondroitin sulphate standards .

Materials and methods

1 . ES was added to a portion of the chondroitin sulphate in DMEM stock solution

to produce a new chondroitin sulphate in DMEM + 1 5 % ES stock solution.

This stock solution was then serially diluted with DMEM + ES to produce

standard solutions containing the equivalent of 0-85 J.Lg mL·1 galactosamine in

DMEM + ES .

2. A second set of water based standards were prepared by dissolving 0 . 1 70 g of

chondroitin sulphate in a 1 5 % aqueous solution of ES ( 1 5 % aqES) to a final

volume of 1 00 mL. This stock solution was then serially diluted with 1 5 %

86

aqES to produce standard solutions containing the equivalent of 0-62 J.Lg mL·'

galactosamine in 1 5 % aqES .

3 . From these standard solutions 1 mL amounts were transferred to microcentrifuge

v ials containing 0.5 mL of 1 2 % TCA, which were then kept at 4 · C for two

hours , before being centrifuged at 1 900 g for 5 minutes .

4. The supernatants ( 1 mL amounts) were dialysed for 36 hours at 4 · C against two

changes of water, each volume of water being the equivalent of 100 times the

combined volume being dialysed .

5. The contents of the dialysis sacks were emptied into cal ibrated vials , had their

volumes standardised with water to 1 . 5 mL, then 1 .4 mL were transferred into

pyrex vials containing 0. 7 mL 6N HCI . These vials were sealed and kept at

1 00 " C for 1 2 hours before 0.6 mL amounts were assayed in tripl icate .

Results and Discussion

Each 0 .6 mL amount final ly assayed was equivalent to 0. 1 78 of the original ammo

sugar concentration of the standard . There was l ittle difference between the chondroitin

sulphate in DMEM and the chondroitin sulphate in 1 5 % aqES standard curves (Figure

2 . 22) . Both sets of standards produced similar sized and shaped absorption spectra to

the previously assayed chondroitin sulphate standards (Figures 2 . 23 & 2 . 24) . Protein

precipitation by TCA, followed by dialysis, was effective in removing most of the

interference associated with the serum supplemented culture medium from the assay of

the amino sugar content of chondroitin sulphate standards . The small associated

reduction in amino sugar yield is within acceptable l imits and appears proportional to

the concentration of the chondroitin sulphate being assayed . Chondroitin sulphate

standards were used in this experiment because proteoglycan standards were not readily

available commercially . Although this method appeared acceptable for assaying

chondroitin sulphate from serum supplemented culture medium it was not known if the

much larger and complex proteoglycan molecules would behave in a similar fashion.

87

0 . 5

•

0 . 4 •

0 . 2 / '

/ o / "

0 . 1 � A/ ' ; �

�

·./ /

0 . 0

0 3 6 9 1 2 1 5 1 8

Galactosamine e quival ent

Figure 2.22 Effect o f serum protein precipitation on the amino sugar

concentration of chondroitin sulphate standards.

Equine serum (ES) supplemented chondroitin sulphate standards disolved in e ither water

or DMEM had their protein component precipitated with trichloroacetic acid before

being dialysed, oven digested in 2N HCl , and assayed for their amino sugar content.

• Chondroitin sulphate in water + ES

v Chondroitin sulphate in DMEM + ES

Q) () � � .0 ... 0 fll .0 <

ii � - .f U: - - � - + - - J _IT- I ±t-' + H- t I _ _ - �- f _

i- - I J .J -+ 1-j I_ j_ I.JJ :h _ _ _ - I _1_1-+++·HI-+1_+-H_ �r=JJ- �- · - - -

-t 7- �- . - - - ,-

J , 1 l+ttHH++HtH-1 Htt tltl+H±J 4 4 0 4 8 0 5 2 0 5 6 0 6 0 0

Wavelength (nm)

88

Equivalent concentration of galactosamine of each pair of absorption spectra : 17 .0 , 12 .7 , 8 .6 , 6 .4 , 4 . 3 , 2 . 1 JLg.

Figure 2.23 Effect of serum protein precipitation and dialysis on the absorption

spectra of aqueous chondroitin sulphate standards.

Q) () � � .0 ... 0 fll .0 <

4 4 0 4 8 0 5 2 0 5 6 0

Wavelength (n m)

6 0 0

Equivalent concentration of galactosamine of each pair of absorption spectra : 1 5 .4 , 1 1 . 5 , 7 . 7 , 5 .7 , 3 . 8 , 1 .9 p.g .

Figure 2 .24 Effect of serum protein precipitation and dialysis on the absorption

spectra of chondroitin sulphate standards in D'MEM.

4 . Separation o f proteoglycans from solutions containing serum proteins

Experiment 1 .

89

The aims of this experiment were to determine if either 2 or 4 % concentrations of TCA

resulted in the precipitation of proteoglycans from solutions (a) containing ES , or (b) not containing ES .

Materials and methods

1 . Strips of articular cartilage were harvested from the intercarpal joints of a

freshly euthanased 8 month old thoroughbred filly and incubated at 37 " C in

sterile water for 48 hours (approximately 6 mg of cartilage mL·1 water) .

2 . Portions of the resulting crude proteoglycan solution had water o r serum added,

corresponding to 1 5 % of the final volume of each solution, to form a

proteoglycan (PG) stock, and a proteoglycan + serum (PG + ES) stock.

3. From the PG stock solution 1 8 x 1 mL aliquots were transferred into three

groups of six microcentrifuge vials. To the first group 0.2 mL of water was

added, to the second group 0.2 mL of 12% TCA was added, and to the third

group 0 .2 mL of 24% TCA was added, resulting in solutions with final TCA

concentrations of 0, 2, and 4 % respectively .

4. From the PG + ES stock solution 12 x 1 mL aliquots were transferred into two

groups of six microcentrifuge vials. To the first group 0 .2 mL of 1 2% TCA

was added, and to the second 0.2 mL of 24% TCA was added

5. Each v ial was kept at 4 ·c for 2 hours, centrifuged at 1 5000 g for 5 minutes ,

then 1 mL of supematant transferred into a dialysis sack and dialysed for 24

hours against two changes of 100 equivalent volumes of chilled de ionised water.

6. The contents of the dialysis sacks were emptied into calibrated vials , had their

volumes standardised with water to 1 .5 mL, then 1 .4 mL were transferred into

pyrex v ials containing 0. 7 mL 6N HCl. The vials were sealed and kept at

lOO" C for 1 2 hours before 0.6 mL amounts were assayed in triplicate for amino

sugar content.

90

Results and Discussion

The average absorbances of the samples from the PG stock solution treated with 2 and

4 % TCA were similar to that of the PG control group. However, the average

absorbances of the samples from the PG + ES stock solution treated with either 2 or 4 %

TCA were markedly different from that of the control group (Table 2 .3 ) .

The results indicated that TCA does not directly precipitate proteoglycans from aqueous

solutions , but that the use of TCA to precipitate ES proteins can result in a proportion

of the proteoglycan molecules being coprecipitated. Extreme pH changes , caused by

strong acids such as TCA, cause denaturation of proteins resulting in the formation of

random coil structures. In solution these random coils become entangled with each

other causing large aggregates and ultimately resulting in precipitation. It is l ikely

some of the proteoglycan molecules also became entangled in these large precipitated

aggregates. However, neither the factors influencing the extent of proteoglycan

coprecipitation, nor the characteristics of the proteoglycan solutions to be assayed are

known. The continued use of TCA to precipitate serum proteins was thus likely to

result in variable and unpredictable amino sugar assay results .

Solution

PG Control

PG + 2% TCA

PG + 4% TCA

PG + ES + 2% TCA

PG + ES + 4% TCA

n

6

6

6

6

6

�ean Absorbance

0. 105

0. 102

0. 106

0.061

0.051

SD

0.006

0.002

0.004

0.006

0.002

Table 2.3 Amino sugar assay absorbances following the addition of TCA and

dialysis (524 nm).

91

Experiment 2.

Because of the coprecipitation problems associated with the use of TCA other

precipitation methods were investigated. Precipitation by increasing the ionic strength

of a solution is known as salting out. Salting out is dependant on the existence of

groups with hydrophobic characteristics on the surface of proteins . When the protein

is in solution the water molecules are forced into close contact with the hydrophobic

groups , isolating them from attracting other similar groups . When salts are added to

the solution water molecules solvate these ions and as the salt concentration increases

water molecules are increasingly drawn away from the hydrophobic groups on the

surface of the protein molecules leaving them free to aggregate with the hydrophobic

groups on other protein molecules .

Proteoglycans are highly charged molecules consisting of approximately 10% protein

and 90% glycosaminoglycans , with the glycosaminoglycans contributing most of the

charged groups . The protein component forms the central core of the molecule from

which the glycosaminoglycan side chains radiate. The solubility of the proteoglycan

molecules would thus be less l ikely to be affected by increasing salt concentrations than

would the solubility of the serum proteins .

The aim of this experiment was to investigate whether the use of ammonium sulphate

to precipitate the serum protein component of the solutions would affect the

proteoglycan concentration of the solution or the assay of its amino sugar component.

Materials and methods

1 . A proteoglycan (PG) stock solution and a proteoglycan stock solution with 1 5 %

added serum (PG+ ES) were prepared in a similar fashion to those prepared in

experiment one.

2. Saturated ammonium sulphate solution (0.5 mL) was added to six

microcentrifuge vials containing 1 mL of the PG stock solution and to six v ials

containing 1 mL of the PG +ES stock solution. Water (0. 5 mL) was added to

92

an additional twelve vials which contained just 1 mL of the PG stock

solution.

3. All vials were left at room temperature for 4 hours before being centrifuged at

1 5000 g for 5 minutes . Six of the twelve vials containing PG and water had 1

mL removed and stored at 4 · C . All of the other vials had 1 mL transferred into

dialysis sacks, which were dialysed for 24 hours against two changes of 100

volumes of chilled ( 4 · C) deionised water.

4. The contents of the dialysis sacks, and the stored PG + water samples , were

emptied into calibrated vials , had their volumes standardised with water to 2 .0

mL, then 1 . 8 mL were transferred into pyrex vials containing 0 .9 mL 6N HCI .

These vials were sealed and kept at 1 oo · c for 12 hours , before 0.6 mL amounts

were assayed in triplicate for amino sugar content.

Results and Discussion

The high osmotic potential created by the addition of the ammonium sulphate resulted

in a net increase in the volume within each dialysis sack. Ammonium sulphate in

combination with dialysis was effective in prevented the development of a black

precipitate during the subsequent acid digestion. Dialysis did not greatly affect the

yield of amino sugars from the PG solutions. The addition of ammonium sulphate to

the PG solutions prior to dialysis made no difference to the absorbances recorded from

the assay , although the final amino sugar concentration of the proteoglycan solution

subjected to these procedures was close to the lowest sensitivity of the assay .

However, the addition of ammonium sulphate to the PG + ES solutions resulted in mean

absorbances approximately 10 fold higher than the other three groups (Table 2 .4) .

93

Solution n Mean SD

Non-dialysed PG Control 6 0 .054 0.003

Dialysed PG Control 6 0 .050 0 .005

PG + NH4S04 6 0.056 0.004

PG + ES + NH4S04 6 0.620 0 .029

Table 2.4 Amino sugar assay absorbances (524 run) following the addition of

NH4S04 and dialysis.

5. Summary

The concentrations of papain used were insufficient to hydrolyse enough of the serum

proteins to prevent the formation of a black precipitate on subsequent acid digestion.

Furthermore , even the lowest concentrations of papain directly reacted with the assay

to produce an absorbance at 524 nm capable of causing interference .

The use of TCA to precipitate the serum proteins caused a coprecipitation of an

unpredictable proportion of the proteoglycan content from the solution. Ammonium

sulphate precipitated sufficient serum protein from solution to prevent the formation of

a black precipitate on subsequent acid digestion. However, its use resulted m absorbances at 524 nm , which were unrelated to the known amino sugar content.

The solubility of a protein molecule in an aqueous solution is determined by the

d istribution of charged hydrophil lic and hydrophobic groups on the surface of the

protein. Protein precipitates are formed by the aggregation of protein molecules and

can be induced by changes in pH, changes in ionic strength, the addition of organic

miscible solvents, or the addition of other inert solutes or polymers . Temperature may

also affect the degree of aggregation achieved.

Proteins can be precipitated out of solution by the addition of water miscible organic

solvents such as acetone and ethanol . Addition of organic solvent lowers the dielectric

94

constant of the solution and hence its solvating power. As a consequence aggregation

of protein molecules through electrostatic attraction occurs. This occurs more readily

when the protein molecules are large and when the pH is close to the isoelectric point

of the protein. The use of water miscible solvents was not attempted as the method is

also often used to precipitate proteoglycans out of solutions .

Precipitation of a protein from a solution can also be induced by adjusting the pH of

the solution to close to the isoelectric point of that protein. At the isoelectric point the

number of negatively charged groups equals the number of positively charged groups

and aggregation of protein molecules occurs due to electrostatic attractions . Culture

medium contains buffers against changes in acidity . The buffering capacity is altered

by the surrounding gaseous environment, the amount and type of metabolic products

produced by the cultured tissue, and the concentration and type of drug added to the

culture medium. Precipitating the serum proteins by adjusting the pH to the isoelectric

point of serum albumin was also considered not feasible as it would have involved

having to titrate each and every culture medium sample to a defined pH .

A variety of chromatographic techniques were investigated . However, equipment

availabil ity , cost, and technical problems, associated with processing several hundred

samples of small volume and very low concentration without diluting each sample below

the minimum sensitivity of the amino sugar assay precluded use of most of these

techniques.

Serum was required in the culture medium for several reasons . Serum has been shown

to contain a number of ' factors' important for the maintenance of tissue health and

metabolic activity during culture. In some media the use of serum has been replaced

with the addition of known quantities of albumin and growth factors, but this was not

an option in the current work because the above experiments suggested albumin was a

major component of the precipitate that formed . Synovial fluid is a highly viscous

dialysate of plasma consisting of approximately 25 mg of protein mL" 1 (Korenek et al. , 1 992) . If the model was to represent the effects of synovial fluid concentrations of

95

certain drugs on articular cartilage, then a significant protein concentration was required

in the culture medium.

The measurement of proteoglycans released into the culture medium was dropped from

the proposed experimental protocol at this point. Many complex sample manipulations

would have been required to remove media, serum, and drug interference from the

assay of the amino sugar content of the proteoglycans. Not only would these

procedures have been costly with respect to the time and resources required, but the

s ize of the combined experimental errors associated with the number of sample

manipulations would have also greatly reduced the value of the results.

C. EXPLANT CULTURE CHARACTERISTICS

1 . Variation i n the amino sugar content and rate of 35S i�corporation

between explants from different sites

96

The number of samples needed to demonstrate a difference between two groups depends

on both the size of the difference and the variance of the population. Because only

small differences between treatment groups were expected the variances associated with

the initial amino sugar and cellular contents of the explants needed to be as small as

possible for sample numbers to be kept within manageable l imits . The variability of

the amino sugar content, and rate of 35S incorporation, within and between s ites in the

middle carpal joints of a single horse were thus determined .

Materials and methods

1. Two hours after the barbiturate euthanasia of an 18 month old thoroughbred

colt, both carpi were skinned and disarticulated at the middle carpal joint . The

cartilage chisel was used to harvest 4 mm wide full thickness strips of articular

cartilage from the left and right middle carpal joint surface of the radial , third ,

and intermediate carpal bones . The cartilage strips from these six sites were

placed in separate screw-capped jars containing chilled ( 4 · C) DMEM (pH 7 .4)

and PSK.

2. Ten explants of articular cartilage (3 mm diameter) were cut from each of these

six sites , and were individually blotted dry , weighed , and placed in 0 . 5 mL of

DMEM + ES (pH 7 .4). All explants were incubated at 3 T C in a humidified

atmosphere consisting of 5 % carbon dioxide and air, with the culture medium

changed and replaced with DMEM + ES supplemented with 5 J.LCi mL-1 Na}5S04

after 48 hours. Each explant was cultured for a further 24 hours before being

subjected to the three wash procedure as described earlier.

3. Each explant was placed in a sealed pyrex vial conta ining 5 mL 2N HCl , kept

at 100 · c for 1 2 hours, then duplicate 0.6 mL amounts were assayed for amino

sugar content, and 1 mL amounts were mixed with emulsion scintillation

97

mixture in duplicate and placed in a scintillation counter. Amino sugar mg-1 wet

weight and dpm mg-1 wet weight values for each explant were then calculated \

by the methods previously described .

Results and Discussion

For each explant, the amino sugar assay was used as a measure of the proteoglycan

content, while the incorporation of 35S , as reflected by the calculated dpm values, was

used as a relative measure of both cellular content and synthetic activity . The between

explant variability in amino sugar content (Table 2 .5 ) and incorporation of 35S (Table

2 . 6) was least in the group of explants from the third carpal bones. However, relatively

large within site variation existed in all of the sites. Generally , sites which had a large

range in explant weights also tended to have more variable results , implying

standardising by weight was not accounting for much of the between explant variation.

Possible reasons for this included; (a) inaccuracies in weight determination due to

variations between explants in surface wetness at weighing, (b) differences in explant

composition caused by variations in sampling depths between explants , and (c) inherent

within s ite variations in amino sugar content and cellularity .

The results indicate that ideally the use of explants from a single site is preferable, but

where greater explant numbers are needed the use of the same s ite in both legs may still

be acceptable . Care must be taken to ensure explants are of a similar dryness, weight,

and sample depth if the variation between explants is to be kept to a minimum. The

use of a DNA assay to standardise each explant may further reduce the variability

between explants due to differences in cellularity.

Carpal bone

Radial

Radial

Third

Third

Intermediate

Intermediate

Side

L

R

L

R

L

R

Explants (n)

9

10

10

10

10

10

Weight ± SD , (mg)

4 .64 ± 0 .77

4 . 60 ± 1 . 13

. 5 .69 ± 0 .35

4 . 83 ± 0 .63

5 . 36 ± 0.78

5 . 62 ± 1 . 1 8

CV (%)

17

25

6

13

15

21

98

AS ± SD CV (JLg mg-1) (%)

1 3 . 30 ± 2 . 6 1 20

1 2 . 02 ± 3 . 98 33

1 3 . 52 ± 2 . 09 1 6

1 4 . 84 ± 1 .96 1 3

1 2 . 73 ± 4 .55 36

14 .23 ± 6 .04 42

Table 2.5 Variability in the amino sugar content (AS) of cultured explants from different carpal bones (JLg mg-1)

Carpal bone

Radial

Radial

Third

Third

Intermediate

Intermediate

Side

L

R

L

R

L

R

Explants (n)

9

10

1 0

1 0

1 0

1 0

Weight ± SD (m g)

4 . 64 ± 0 .77

4 .60 ± 1 . 1 3

5 . 69 ±0.35

4 . 83 ± 0 .63

5 . 36 ± 0 .78

5 . 62 ± 1 . 1 8

CV (%)

17

25

6

1 3

1 5

2 1

1 6440 ± 4 1 25

1 5790 ± 6890

1 4490 ± 3475

1 1 275 ± 2000

1 42 1 5 ± 2790

13710 ± 4605

CV (%)

25

43

24

1 7

1 9

3 3

Table 2.6 Variability in the incorporation of 35S by cultured explants from different carpal bones (dpm mg-1)

99

2. Viability of chondrocytes in cultured explants of equine articular cartilage

The aim of this experiment was to determine the effect of tissue culture duration on the

viability of chondrocytes in explants of equine carpal articular cartilage.

Materials and methods

1 . Two hours after the barbiturate euthanasia of a five year old thoroughbred

gelding , both carpi were skinned and disarticulated at the middle carpal joint.

A razor blade was used to harvest full thickness strips of articular cartilage from

the exposed carpal bones and the strips were placed in a screw-capped jar

containing chilled (4 " C) DMEM (pH 7 .4) and PSK. A cartilage punch was then

used to cut 30 x 3 mm explants from these strips, and each explant was

randomly allocated to one of six groups until each group contained five explants .

2 . The first group of five explants was immediately fixed in glutaraldehyde (2 . 5 %

concentration in phosphate buffer at pH 7 .2) for 6 hours, rinsed in three changes

of PBS, and stored in 70% ethanol . The remaining 25 explants were placed in

individual tissue culture wells containing 1 .0 mL of DMEM + ES (supplemented

with 100 IU m.L-1 penicillin and 100 p.g mL-1 streptomycin) and were · incubated

at 37 · C in a humidified atmosphere of 5 % carbon dioxide and air .

3 . The culture medium for each explant was changed on days 1 , 2 , 4 , 6 , and 8 .

A t each change one group of five explants was removed from culture and fixed

in glutaraldehyde, rinsed in PBS, and stored in 70% ethanol .

4. Each of the fixed explants was cut in half, and one half was processed and

embedded in paraffin wax while the other half was stored. Sections (6p.) across

the full diameter of each disc were cut and stained with haematoxylin, eosin,

and alcian blue (pH 7 . 2) .

5 . The total number of nucleated cells i n each section was counted at 400 x

magnification, followed by a count of the number of dead cells under an oil

immersion lens at 1000 x magnification. Cell death was assessed purely on

nuclear morphology . Dead cells were defined as ' nucleated cells in which the

nuclear material had become pyknotic i .e . the nucleus had shrunk in size and the

100

chromatin had condensed into a solid dark coloured structureless mass or

masses ' . Empty lacunae and anuclear cells were not included in either the total \

or the dead cell counts as neither the orientation of the cell nor the effects of

processing could be excluded as factors involved in their appearance .

Results and Discussion

The results are shown in Table 2. 7 and Figure 2.25 . Due to difficulties associated with

the use of a folded razor blade to harvest strips of cartilage, not all explants were of the

same thickness, and the thickness of some ex plants also varied across their diameter.

In addition, because of possible contamination during explant processing 100 IU mL1 penicillin and 100 Jlg mL·' streptomycin were used during culture.

concentrations are routinely used during cell culture .

These

There was an initial increase in the average number of dead cells in the explants after

the first day of tissue culture, followed by a more gradual increase over the next 7

days . The average viability of the chondrocytes exceeded 90% for the first 8 days of

culture . However, the variability found between sections, generally increased with

culture duration. Chondrocyte death was greatest at the ends of each section, where the

punch had cut perpendicular to the articular surface (Figure 2 . 26) , followed by

chondrocytes in the tangential zone where cell density was greatest (Figure 2 . 27) .

Chondrocyte death was least noticeable in the radiate and calcified zones of each

section. Cell death at the cut outside edges of the cultured explants was expected .

However, the high proportion of cell deaths occurring in the morphologically intact

tangential zone in comparison to the radiate and severed calcified zones was not .

Cell health was generally thought to decline during the culture period, as indicated by

an increasing prevalence of cytoplasmic vacuolation, anuclear cells, and variable

cellular staining characteristics (Figure 2 .28). Wide variation in cellular density ,

cellular distribution characteristics, and width of samples was observed . A more

standardised sampling procedure, from a smaller number of morphologically similar

sites , may help reduce the variation between explants .

101

Classification of cell death purely on the presence of a pyknotic nucleus may have

underestimated the true percentage of dead chondrocytes, but was considered the most \

clear-cut and objective assessment. Never-the-less , the study demonstrated that a high

percentage of the chondrocytes within tissue cultured explants of equine middle carpal

joint articular cartilage can be maintained viable for periods of at least 8 days .

Explant Non-cultured Days cultured

controls 1 2 4 6 8

1 0/465 22/436 19/4 10 23/559 34/490 69/398

2 0/449 1 8/467 26/560 34/345 33/450 1 2/844

3 0/437 1 6/397 14/758 45/491 74/761 20/461

4 0/442 24/457 23/435 24/5 1 5 54/469 70/56 1

5 0/403 20/440 5/55 1 9/41 3 40/563 10/357

Dead 0 1 00 87 1 35 138 1 8 1

Total 2 1 96 2 1 99 27 1 4 2323 2733 261 7

% Dead 0 4 . 5 3 .5 6 . 0 8 . 1 7 . 7

% Viable 1 00 95 . 5 96. 5 94. 0 9 1 .9 92. 3

SD 0 0 . 6 2 . 0 3 . 3 2 . 8 7 . 0

Table 2 . 7 Chondrocyte death relative to time cultured (dead/total) .

102

1 1 0 E r r o r B a rs = S D 1 0 5

1 0 0

9 5

Cl) 9 0 -..c ctl 8 5 ...... > I'll 8 0 Cl) � >.

7 5 () 0 � 'd 7 0 � 0 6 5 ..c: u

� 6 0

5 5

5 0

4 5

4 0

0 1 2 3 4 5 6 7 8

Culture duration ( d a y s )

Figure 2.25 Effect of culture duration on the viability of chondrocytes within

explants of equine articular cartilage.

103

./ ' •

•

•

•

•

•

Figure 2.26 Photomicrograph showing cell death at the edge (J') of a cultured

explant of equine carpal articular cartilage (400x magnification) .

104

•

•

• - •

• f •

•

•

Figure 2.27 Photomicrograph showing cell death at the articular surface (J') of

a cultured explant of equine carpal articular cartilage (lOOOx

manification) .

105

Figure 2.28 Photomicrograph showing cell vaculation (/') in a cultured explant of equine carpal articular cartilage ( 4000x magnification) .

106

3. Variation in the biosynthetic rate and total amino sugar concentration

relative to time cultured

The aims of this experiment were (a) to compare the use of a DNA assay to the use of

wet weight as a means for standardising both the amino sugar content and incorporation

of 35S by each explant, and (b) to determine how the amino sugar content and

incorporation of 35S varied with culture duration .

Materials and methods

1 . Two hours after the barbiturate euthanasia of an 9 year old thoroughbred

gelding, both carpi were skinned and disarticulated at the middle carpal joint .

The cartilage chisel was used to harvest full thickness 4 mm wide strips of

articular cartilage from the right and left third and radial carpal bones which

were placed in a single screw-capped jar containing DMEM + PSK at 4 · C .

From these strips 80 x 3 mm diameter explants were punched , individually

blotted dry , weighed, and randomly allocated into one of eight groups until each

group contained ten explants.

2. The explants from group one were placed in individual vials and frozen (-20 . C) .

Explants from group two were placed into culture plate wells containing 0 .5 mL

of DMEM + ES (pH 7 .4) and 5 J.LCi mL-1 Na/5S04 • The explants from groups

three to eight were placed into culture plate wells which contained 0 .5 mL

D MEM + ES . All groups except group one were then incubated at 3 7 · C in a

humidified atmosphere of 5 % carbon dioxide and air.

3. After 24 hours of culture the explants from group two were harvested , washed

using the previously described three wash procedure , and placed in individual

vials and frozen.

4. After 1 , 2 , 3, and 4 days of culture groups 3, 4, 5, and 6 respectively were

placed in radiolabelled culture medium for their final 24 hours of culture before

also being harvested, washed and frozen. The culture media of all groups not

being radio labelled were replaced with fresh DMEM + ES every 48 hours .

107

5. All frozen samples were thawed, had 1 mL of PBS + 50 Jll of papain24 added,

and were placed in a water bath at 65 · C for 24 hours . \

6. Calf thymus DNA standards, ranging from 0 to 10 Jlg DNA mL·1 , were

prepared as previously described . From each of the standard solutions, and

from each of the papain digested samples, 0.2 mL amounts were transferred into

1 mL polystyrene microcentrifuge tubes , placed in crushed ice and briefly

sonnicated . The DNA content of 50 J.Ll amounts were assayed in duplicate

according to the method of Labarca and Paigen ( 1980) using a 0 . 1 llg mL·1

concentration of the Hoechst 33258 reagent solution.

6 . The remaining 0 .8 mL of each o f the papain digested samples was mixed with

3 . 2 mL of 2 . 5N HCI , three papain blanks were prepared by mixing 50 p.l of

papain with 5 mL of 2N HCI , and a series of galactosamine standards in 2N

HCI were also prepared . All vials were sealed and kept at 1oo· c for 12 hours

before 0 .6 mL amounts of each were assayed in triplicate for amino sugar

content, and 1 .0 mL amounts of each had their dpm determined in duplicate, by

the previously described method .

Results and Discussion

The assay of the DNA content of each explant necessitated a number of extra sample

manipulations , including the use of a papain digest, which potentially reduced the

accuracy of the assay of the amino sugar content of each explant. The concentration

of papain in the papain blanks was equivalent to the papain concentration in each

sample. The average absorbance (0.084) produced by the assayed papain blanks was

subtracted from each of the assayed samples before amino sugar content calculation.

There was no apparent advantage gained by using the fmal DNA content of each

explant to standardise its 35S incorporation and amino sugar content (coefficients of

variation = 39 and 26% ) , compared to the use of the wet weight of each explant

(coefficients of variation = 42 & 19%) , (Figures 2 .29-2 .32 & Appendix Tables 8-1 1 ) .

The aim of standardising each explant was to reduce the variation in the initial amino

24 Stabilised liquid papain, Salmond Smith Biolab N.Z. Ltd.

3 0 --.

2 5 bO E ..........

2 0 bO ::l -

...., 1 5 ..c: bO ..... 1 0 11)

11= .......... rr.J 5 < 0

0 1 2 3 4 5

Error b ars = SO

6

A � 1 9.9 B ; -0 78 r ; 0 . 73

7

Culture duration ( d ays)

108

Figure 2.29 Effect of culture duration on amino sugar content of explants relative

to their initial wet weight.

4 0

f 3 5

-. tlD 3 0 ::l .......... 2 5 tlD ::l

-2 0 < z 1 5 Q .......... 1 0 rr.J <

5

0

0 1 2

. . . . . . . . . . . . . . . . . . . . . . . . . . ! l

3 4

Error bars = SD

5 6 7

Culture durati o n ( days )

Figure 2 .30 Effect of culture duration on amino sugar content of explants relative

to their fmal DNA content.

\

� :��� � s 3 0 0 0 r � 2 5 0 0 till

..... 2 0 0 0 Cl> l!:= ......... s p. "'CC

1 5 0 0

1 0 0 0

5 0 0

109

Error bars = SD

0 �------�--------�------�--------�------� 0 1 2 3 4 5

Culture duratio n ( days )

Figure 2 .31 Effect of culture duration on 35S incorporation by explants relative

to their initial wet weight.

4 0 0 0 r

3 5 0 0 I

- 3 0 0 0 till :::1 2 5 0 0 .......... < z 2 0 0 0 0 .........

s 1 5 0 0

E rror bars = SD 1 1

p. "'CC 1 0 0 0

5 0 0

0

0 1 2 3 4 5

Culture duratio n ( days)

Figure 2.32 Effect of culture duration on 35S incorporation by explants relative

to their fmal DNA content.

110

sugar content and cellularity of the explants. How accurately the final DNA content

reflects variations in the initial cellular content of the explants is not known. Cytotoxic \

effects associated with culture and different drug concentrations could also be expected

to have variable effects on the final DNA content of each explant. For these reasons,

and continuing problems with repeatability , the assay of DNA content was excluded

from the proposed experimental protocol .

The amino sugar/weight results indicate cultured explants of equine carpal articular

cartilage have a net loss of proteoglycans during tissue culture , with the final

proteoglycan content tending to be inversely related to culture duration. In contrast the

average rate of proteoglycan synthesis initially increased up to the end of the third day

of culture, then remained relatively constant for a period. Delaying both the addition

of the drugs to be tested and the initiation of radiolabell ing until after the explants had

acclimatised to the in vitro conditions would thus be more likely to expose concentration

related drug effects on proteoglycan synthesis.

MATERIALS AND METHODS: SECTION TWO

USE OF THE MODEL

A. EXPLANT CULTURE

1. Source of articular cartilage

111

Articular cartilage was harvested from the middle carpal joints of 12 thoroughbred or

thoroughbred cross horses. These ranged in age from 2 to 12 years and (a) had been

presented at the Massey University Equine Clinic and euthanased by barbiturate

overdose for reasons unrelated to, and unlikely to affect, their carpal articular

cartilages, or (b) were horses from a local hunt club or ab�ttoir which had been killed

by a bullet or captive bolt penetrating the brain.

All animals were free from chronic metabolically debilitating disease, and had no

history of corticosteroid, NSAID, or other recent drug therapy . Each middle carpal

joint was inspected visually, and any joint with abnormal cartilage at or close to the

sites of harvest was discarded .

2. Preparation of cartilage samples

Within 6 hours of the death of each horse, both carpi were skinned and disarticulated

at the middle carpal joint. The custom made cartilage chisel was used to harvest 4 mm wide full thickness strips of articular cartilage from the third and radial carpal bones

(Figure 3 . 1 ) . The cartilage from each site was placed in a separate screw-capped jar

containing chilled ( 4 · C) DMEM (pH 7 .4) and PSK. A punch was used to cut as many

3 mm diameter explants as possible from the strips of cartilage from each site Figure

3 . 2 and 3 . 3). The explants from the third carpal bones were processed first. Each was

individually blotted dry and weighed on a Mettler balance (reproducibility 0.05 mg) ,

then placed in 1 .0 mL culture plate wells containing 0.5 mL DMEM (pH 7 .4)

1 12

Figure 3 . 1 Photograph o f an open equine middle carpal j oint showing the sites

(top = third carpal bone, bottom = radial carpal bone) where the

strips of articular cartilage were harvested from.

I

Figure 3.2 Strip of articular artilage from which explants have been cut.

1 13

Figure 3.3 Explants suspended in chilled DMEM awaiting further processing.

114

supplemented with 1 5 % heat inactivated equine serum (ES). The explants rested

directly on the well bottoms and no attempt was made to influence which surface faced

uppermost. Explants were then incubated at 37 · C in a humidified atmosphere of 5 %

C02 and air.

S ix explants from the radial carpal bones were randomly selected (non-cultured control

group) and fixed in Bouins fluid for 16 to 24 hours, then transferred to 70% ethanol .

The rest of the explants from the radial carpal bones were placed in 1 .0 mL culture

plate wells containing 0.5 mL DMEM + ES (pH 7 .4) and incubated with the third

carpal bone explants .

Both sets of explants were culture.d for an initial 48 hour stabilising period with their

media replaced after 24 hours .

3. Depo-Medrol (MPA) trial

Explants of articular cartilage from six horses were used in this study . A series of

suspensions containing 0, 0.4, 4, and 40 mg mL1 of methylprednisolone acetate were

prepared fresh each day by mixing variable amounts of Depo-Medrol25 and PBS .

The 48 hour explant cultures from each site were randomly allocated into four

control/treatment groups , until each group contained 25 % of the total number of

explants . Each explant was maintained for a further 72 hours in DMEM + ES (0 . 5 mL)

to which either 0 .025 mL of PBS (the controls) or 0.025 mL of one of the MPA

suspensions (the treatment groups) had been added . Media and PBS/MPA were

changed at 24 hour intervals. The medium used on the explants from the third carpal

bones for the last 24 hours of culture (96- 120 hours) was additionally supplemented

with 5 J.LCi mL·1 Na/5S04•

25 Depo-Medrol , 40 mg ml·1 methylprednisolone acetate, Upjohn Inter-American Corporation.

4. Phenylbutazone trial

1 1 5

Explants of articular cartilage from six different horses were used in this study .

Phenylbutazone26 (PBZ) was dissolved in 2M NaOH and added to DMEM during its

preparation to form a DMEM + PBZ stock solution (pH 7 .4) . This stock solution was

serially diluted with DMEM, and then had ES added, to give concentrations of 0, 2 ,

20, 200, and 2000 J.Lg mL-1 PBZ in DMEM + ES(15 %) .

Due to inadequate sample numbers, the explants allocated to the 2000 J.Lg mL-1

concentration of PBZ came from only five of the six horses for the histological study

(radial carpal bones) , and four out of the six horses for the amino sugar and 35S

incorporation studies (third carpal bones) . In addition, four of the five 2000 J.Lg mL-1

treatment groups in the histological study contained one less explant than the other

groups . These constraints withstanding , the 48 hour explant cultures from each site

were randomly allocated into either four or five control/treatment groups, until each

group contained 25 % or 20% respectively of the total number of explants . Each group

of explants was then maintained for a further 72 hours in culture medium

(DMEM + ES) containing 0, 2, 20, 200, or 2000 J.Lg mL·1 PBZ, which was changed

every 24 hours . The media used for the explants from the third carpal bones for the

last 24 hours of culture (96- 1 20 hours) were additionally supplemented with 5 J.LCi mL-1

Na/5S04 • Any explants from culture plate wells that showed any evidence of bacterial

contamination were discarded.

B. POST-CULTURE PROCESSING

1 . Explants from the radial carpal bones

Immediately after culture termination the explants from the radial carpal bones were

prepared for histological examination. Each group was fixed in Bouins fluid for 1 6 to

24 hours, then transferred to a solution of 70% ethanol in water and stored .

26 Phenylbutazone, gift from Ethical Agents Australia Ltd.

2. Explants from the third carpal bones

116

The explants from the third carpal bones were prepared for biochemical analysis . Each

explant was subjected to the three wash procedure (as described in materials and

methods : section one) , and was then placed in a 5 mL pyrex screw-top vial containing

5 mL of 2N HCl, tightly sealed with a teflon l ined screw-cap, and incubated at 100 · C

for 1 6 hours . The contents of any vials which had obviously leaked during the acid

d igest were discarded.

C . ANALYTICAL TECHNIQUES

The amino sugar content of each explant was assayed as a measure of its

glycosaminoglycan and proteoglycan content. The incorporation of 35S during the _final

24 hours of culture was used as a relative measure of glycosaminoglycan and

proteoglycan synthesis. Histological assessment of cell death was used as measure of

the relative cytotoxicity of the different doses/concentrations of the two drugs used .

1 . Amino sugar assay

The amino sugar content of 0. 6 mL amounts of the acid digested explants were assayed

in duplicate according to the Gatt and Berman ( 1 966) modification of the Elson and

M organ ( 1933) colorimetric method for the determination of amino sugars, as described

in materials and methods : section one . The total amino sugar (AS) content of each

explant was then calculated, divided by the initial wet weight of the explant, and

expressed as 11-g AS mg·1 •

2. Scintillation counting procedure

From each of the acid ·digests 0 .5 mL amounts were subjected to the scintillation

counting procedure in duplicate, as described in materials and methods : section one .

The calculated dpm occurring in each explant was then divided by the initial wet

weight of the explant and expressed as dpm mg·1 •

3 . Histological evaluation

1 1 7

Each o f the fixed explants was cut i n half, and one half was processed and embedded

in paraffm wax while the other half was stored. Sections (6�-t) across the full diameter

of each explant were cut and stained with alcian blue (pH 7 .2), haematoxylin and eosin (H & E) . A blinded assessment of both dead and total cell counts was made by an

independent histologist, using the method described in materials and methods : section

one . The percentage of cells dead in each explant was then estimated from these

counts .

D. STATISTICAL METHODS

1 . Explants from the radial carpal bones

For the explants from each horse, the percentage in each section classified as dead was

calculated, then the mean dead cell percentage of the non-cultured group was subtracted

with any negative results being rounded up to zero . The results from the explants of

all six horses were then combined into their respective control/treatment groups, the

distributions normalised by log transformation, and the means then analysed using

Scheffe ' s pairwise comparisons (a = 0 .05) .

2. Explants from the third carpal bones

The inherent variation in the amino sugar content and incorporation of 35S between the

explants from different horses was controlled by dividing the results of each explant

by the mean result of the control group from the same horse. The results from the

explants of all six horses were then combined into their respective treatment groups.

The distributions of the scintillation results were normalised by square root

transformation. However, some heteroscedasticity still existed. Differences between

treatment groups were analyzed by the method of l inear contrasts (Sokal & Rohlf,

198 1 ) . For each drug and each parameter, three non-orthogonal combinations of

treatment and control groups were used to examine the null hypotheses.

( 1 ) The drug has no effect.

U control = U treatments

118

(2) The drug has no effect at the concentrations found in synovial fluid in vivo.

(3) The effect of the drug is not greater at higher concentrations than found in vivo .

!bol'g mL-1 + u2001'g mL·I = u20001'g mL· I (for MPA) 2

All hypotheses were investigated at the a = 0. 05 level of significance . The second

and third null hypotheses were only investigated if the first was rejected .

RESULTS

A. DEPO-MEDROL (MP A) TRIAL

1 19

Seventeen of the 280 cultured explants from the third carpal bones were excluded from

the trial due to evidence of bacterial contamination of their culture wells ( 4), or

noticeable leakage from their sealed pyrex vials during acid digestion ( 13) . Two of the

1 80 explants from the radial carpal bones were lost during processing .

1 . Effect o f Depo-Medrol on amino sugar content

The dose related effect of Depo-Medrol on the amino sugar content of cultured TCB

explants from six horses is summarised in Figure 4 . 1 . When all the treatment groups

were combined, the mean amino sugar content of the explants cultured in the presence

of Depo-Medrol was higher than that of the cultured controls (F0• 260>, p = 0.017) . The

mean amino sugar content of the combined 20 and 200 /lg mL·1 treatment groups was

also higher than that of the cultured controls , but the difference was not significant (F(l .

195> , p = 0.075) . The mean amino sugar content of the combined 20 and 200 /lg mL-1

treatment groups was also not significantly different from that of the 2000 J.tg mL-1

treatment group (F(l . 2oo> • p = 0 . 1 350) .

2. Effect of Depo-Medrol on 35S incorporation

The dose related effect of Depo-Medrol on the incorporation of 35S by cultured TCB

explants from six horses is summarised in Figure 4.2. Depo-Medrol depressed the

incorporation of 35S in a dose dependant fashion. Incorporation of 35S by the cultured

controls was higher than that by the combined treatment groups (F0• 260>, p < 0.001) ,

and the 20 and 200 J.Lg mL-1 treatment groups (F0. 195>, p < 0.001) . Furthermore the

incorporation of 35S by the combined 20 and 200 /lg mL-1 treatment groups was also

higher than that by the 2000 J.Lg mL-1 treatment group (F0, 200>, p < 0.001) .

120

1 2 0 Error bars -= SEW

1 1 5

1 1 0

* 1 0 5

-0 ... ..,J

1 0 0 r:: 0

u

� 9 5

9 0

8 5

8 0

0 2 0 2 0 0 2 0 0 0

Methylpre dnisolone acetate ( J.Jg ml - 1 )

Figure 4. 1 Effect of Depo-Medrol on the amino sugar content of cultured

explants of equine middle carpal joint articular cartilage.

* The amino sugar mg·1 value of each explant was divided by the mean amino sugar

mg·1 value of the control group from the same horse. The combined results from all

the horses were then separated from the four control/treatment groups, averaged and

expressed as percentages + SEM (number of explants in each group: A = 6 1 , B = 67,

C = 69, and D = 66) .

Groups B, C , and D combined are significantly different from group A (p = 0.017) .

However, group B and C combined are not significantly different from either group A

(p = 0.075) or group D (p = 0. 135).

121

1 2 0 Error bars = SEM

1 0 0

8 0

*

6 0

4 0

2 0 D

0

0 2 0 2 0 0 2 0 0 0

Meth ylprednisolone a c e tate (J.Lg ml- 1 )

Figure 4.2 Effect of Depo-Medrol on 35S incorporation by cultured explants of

equine middle carpal joint articular cartilage.

* The dpm mg·1 value of each explant was divided by the mean dpm mg·1 value from

the same horse. The combined results from the explants of all the horses were then

· separated out into the four control/treatment groups, averaged, and expressed as

percentages + SEM (number of explants in each group: A = 6 1 , B = 67, C = 69,

and D = 66).

Groups B, C , and D combined are significantly different from group A (p < 0.001) .

Groups B and C combined are also significantly different from group A (p < 0.00 1) ,

and are significantly d ifferent from group D (p < 0.001) as well .

3. Effect of Depo-Medrol on chondrocyte viability

122

The dose related effect of Depo-Medrol on the viability of chondrocytes within cultured

RCB explants from s ix horses is summarised in and Figure 4.3 . Depo-Medrol affected

the percentage of pyknotic nuclei found only at the 2000 p.g mL-1 concentration (p <

0 .001) .

123

2 0

Error bars = SE.M 1 8

1 6

*

"t1 1 4 «S Q) "t1 1 2

l1.l Q) ..., 1>. 1 0 C) 0 J..

'Tj 8 1=:

0 ..r:: u 6

�

4

2

0

0 2 0 2 0 0 2 0 0 0

M ethylpre dnisolone acetate (J..Lg ml- 1 )

Figure 4.3 Effect of Depo-Medrol on the viability of chondrocytes within

cultured explants of equine middle carpal joint articular cartilage.

* The percentage of chondrocytes determined as dead in each cultured explant was

reduced by the average percentage of chondrocytes found dead in the non-cultured

group of explants from the same horse. Any resulting negative percentages were

rounded up to zero, then all results were combined into the four control/treatment

groups and averaged (number of explants in each group: 0 = 35, 20 = 35 , 200 = 36,

and 2000 = 36) .

The percentage of chondrocytes dead in the 2000 Jlg ml-1 group was s ignificantly

different from the other groups (p < 0. 001) .

B. PHENYLBUTAZONE (PBZ) TRIAL

124

Fifteen of the TCB explants , out of the 296 cultured, were excluded from the trial due

to evidence of bacterial contamination of the culture media (3) , or noticeable leakage

from the sealed pyrex vials during acid digestion (12) . Because insufficient explants

could be harvested from the radial carpal bones slightly lower numbers of explants were

allocated to the non-cultured group and 2000 llg mL·1 treatment group. One of the 1 85

explants cultured was lost during processing .

1 . Effect of PBZ on amino sugar content

The effect of different concentrations of PBZ on the amino sugar content of cultured

TCB explants from six horses is summarised in Figure 4.4 . When al l the treatment

groups were combined , the mean amino sugar content of the explants cultured in the

presence of PBZ was higher than that of the cultured controls (F0 . 277) , p = 0 .0 1 1 ) .

The mean amino sugar content of the combined 2 and 20 J.Lg mL-1 treatment groups was

also higher than that of the cultured controls (F(I . 183> , p < 0.0 1 ) . However, there was

no significant difference between the amino sugar content of the combined 2 and 20 llg

mL-1 treatment groups and the combined 200 and 2000 llg mL1 treatment groups (F( I .

216) • p = 0.59) .

2. Effect of PBZ on 35S incorporation

The effect of different concentrations of PBZ on the incorporation of 35S by cultured

TCB explants from six horses is summarised in Figure 4 . 5 . Only the 2000 J.Lg mL-1 PBZ treatment group had a significantly lower mean incorporation of 35S than the

control group (F0 . 216> , p < 0 . 00 1 ) .

125

1 2 0 Error bars • SEll

1 1 5

1 1 0

* 1 0 5

..... 0 r... .....,

1 0 0 s:: 0 (.) � 9 5

9 0

8 5

8 0

0 2 2 0 2 0 0 2 0 0 0

Phenylbutaz one (J.�-g ml-1 )

Figure 4.4 Effect of phenylbutazone on the amino sugar content of cultured

explants of equine middle carpal joint articular cartilage.

* The amino sugar mg-1 value of each explant was divided by the mean amino sugar

mg-1 value of the control group from the same horse. The combined results from all

the horses were then separated into four control/treatment groups, averaged and

expressed as percentages ± SEM (number of explants in each group: A = 62, B = 61 ,

C = 62, D = 60, E = 36) .

Groups B, C , D, and E combined are significantly different from group A (p = 0.0 1 1) .

Groups B and C combined are also significantly different from group A (p < 0.001),

but are not significantly different from groups D and E combined (p = 0 . . 59) .

126

1 2 0 Error bars = SEM

*

0 2 2 0 2 0 0 2 0 0 0

Phenylbutazone (f..Lg ml- 1 )

Figure 4.5 Effect of phenylbutazone on 35S incorporation by cultured explants

of equine middle carpal joint articular cartilage.

* The dpm mg·1 value of each explant was divided by the mean dpm mg·1 value of the

control group from the same horse. The combined results from the explants from all

the horses were then separated into five control/treatment groups, averaged and

expressed as percentages + SEM (number of explants in each group : A = 62 , B = 6 1 , C = 62 , D = 60, and D = 36).

Only the 2000 J.Lg ml·1 phenylbutazone treatment group had a significantly lower mean

35S incorporation than the other groups (p < 0.001) .

3. Effect of PBZ on chondrocyte viability

127

The effect of different concentrations of PBZ on the viability of chondrocytes within

cultured RCB explants from six horses is summarised in Figure 4.6. PBZ affected the

percentage of pyknotic nuclei found only at the 2000 J.Lg m.L-1 concentration (p < 0.01) .

128

2 0

Error b a r s = SEM 1 8

1 6

*

"0 1 4 a:l Q) "0 1 2 D'l Q)

.... � 1 0 (.) 0 J...

"0 8 0

0 ..c= t.) 6

�

4

2

0

0 2 2 0 2 0 0 2 0 0 0

Ph enylb utazone (j..Lg ml- 1 )

Figure 4.6 Effect of phenylbutazone on the viability of chondrocytes within

cultured explants of equine middle carpal joint articular cartilage.

* The percentage of chondrocytes determined as dead in each cultured explant was

reduced by the average percentage of chondrocytes found dead in the non-cultured

group of explants from the same horse. Any resulting negative percentages were

rounded up to zero, then all results were combined into the five control/treatment

groups and averaged (number of explants in each group: 0 = 29, 2 = 32, 20 = 32 ,

200 = 32, and 2000 = 28).

Only the 2000 p.g ml-1 phenylbutazone treatment group had a significantly greater mean

percentage of chondrocytes that died during culture than the other groups (p < 0.01 ) .

C. HISTOLOGICAL TRIAL OBSERVATIONS

129

The staining characteristics, chondrocyte morphology, and size of the sections examined

varied widely both within groups of explants from the same horse and between similar

groups of explants from different horses. The appearance of individual cells in any one

section varied substantially with nuclei of many shapes and sizes seen. Empty lacunae

were seen in all sections. In some cases, the missing nuclei appeared to have been

d isplaced into the surrounding matrix during processing (Figure 4. 7) . The intensity of

the haematoxylin staining of the nuclei also varied between similar sections. However,

the nuclei tended to be darker and smaller in the sections exposed to the highest drug

concentrations (Figure 4 . 7) . Cytoplasmic vacuolation was more apparent subjectively

in the sections with higher dead cell percentages . Cell death was most commonly seen

close to the articular surface and at the ends of the sections (Figure 4 . 8) . Only a small

number of closely associated clusters of chondrocytes were seen in any one section, and

no obvious difference in their number was noticed between any of the drug

concentrations and the controls . The clusters seen within the transitional and radiate

zones appeared to be contained within separately delineated ovoid structures (Figure

4 . 9) .

The sections cut from the non-cultured group of explants consistently had the most

intense alcian blue staining . However, even in this group the staining intensity was not

always consistent between sections from the same horse (Figure 4. 10) , and there was

substantial variation between sections from different horses. Sections of the cultured

control group of explants tended to stain least with alcian blue, but no difference could

be observed between the alcian blue staining intensities of explants exposed to the

d ifferent concentrations of either Depo-Medrol or PBZ. In both drug trials

interterritorial staining intensities appeared to be affected before territorial staining

intensities .

130

• ..

Figure 4. 7 Photomicrograph of an empty lacunae (/') with the displaced nucleus

sitting adjacent (lOOOx magnification) .

131

I

Figure 4.8 Photomicrograph of two dead chondrocytes (J') close to the articular

surface ( 4000x magnification) .

132

Figure 4.9 Photomicrograph of a chondrocyte cluster (?) ( 4000x magnification) .

133

• •

•

Figure 4. 10 Comparison of the variation in proteoglycan content, as shown by

alcian blue staining, of two sections cut from non-cultured explants

harvested from the radial carpal bone of the same horse ( 400x

magnification) .

134

DISCUSSION

The objectives of this investigation were to (a) establish and validate a tissue culture

model of equine carpal articular cartilage, and (b) use this model to examine the direct

effects of a series of concentrations/doses of phenylbutazone and a methylprednisolone

formulation on chondrocyte viability, and chondrocyte mediated degradation and

synthesis of matrix proteoglycans .

A. DISCUSSION OF THE METHODS

The rationale for using an in vitro model was based on the assumption that the clinical

signs associated with both disease and drug effects are reflections of actions exerted at

the cellular level . The in vitro maintenance of tissue allows for a more controlled

environment in which the study of specific interactions can be isolated from

confounding variables. Explant culture was used in deference to cell culture because

the metabolic activity of chondrocytes within articular cartilage is to a large extent

controlled by the microenvironment created by the matrix immediately surrounding

each cell . Chondrocytes make up less than 5 % of the composition of articular

cartilage, and their effects on its biomechanical properties are solely mediated through

their effects on the components of the matrix .

Explants of equine articular cartilage have been cultured by a number of other

researchers for durations of up to 13 days (Pool et al. , 1980; Chunekamrai et al. ,

1989; Caron et al. , 1991 ; MacDonald et al. , 1992; Caron et al. , 1993) . The use of

explants maintained many of the normal tissue interactions present in joints, but also

resulted in structurally abnormal surfaces being exposed, and possibly changed normal

nutrient and metabolic gradients . In addition, the large inherent variation between

explants in cellular and proteoglycan content meant large sample numbers were

required to ensure clinically significant treatment differences could be shown. The site

of harvest, and the size and shape of the explants, were chosen to maximise the number

of explants while minimising between-explant variation and the cut surface area to

volume ratio of each explant.

135

The explants came from horses destined for destruction, so strictly speaking they could

not be considered to be representative of the articular cartilage of thoroughbreds in

training. Nevertheless, all joints used were considered healthy and were without any

obvious degenerative changes. Sample allocation was randomised with the exception

that fixed sample sizes were used and the highest concentration group had its number

of explants reduced if any shortage arose. Large sample sizes minimised any bias this

may have had on the results . The conditions from time of death until culture initiation

adversely affect chondrocyte metabolism. Explants were thus allowed to recover and

acclimatise to the in vitro culture conditions for 48 hours before the drugs to be tested

were added. In this way the effects of the drugs were thought to be more

representative of their effects on pathological processes as they might occur in vivo,

rather than of their modifying effects on the stresses associated with culture initiation.

Radiolabelling was initiated only after 96 hours of culture to allow both a stabilisation

of synthetic rates and time for the drugs to have exerted their effects.

Serum was considered an essential part of the culture media as it provided a source of

growth factors, cholinesterases, and albumin. Adult equine serum was used to ensure

the types and concentrations of growth factors present closely resembled those of the

animals from which the explants originated. Growth factors were necessary to

maintain proteoglycan synthesis for the duration of the culture, while cholinesterases

were necessary to convert methylprednisolone acetate (MPA) into the active

methylprednisolone (MP) form. However, it was not known what percentage of the

cholinesterases survived the heat inactivation of the serum. As PBZ is normally

extensively protein bound ( > 98 %), albumin was required to ensure the chondrocytes

were exposed only to the fraction of PBZ that would 'normally' exist in the unbound

form.

The use of serum in the media meant the amino sugar content of the released

proteoglycans could not be assayed by the method of Gatt & Berman (1966) (see

Materials & Methods: Section One) . As a result there was no direct measure of

proteoglycan catabolism. However, the final amino sugar content of each explant

reflected both the anabolic and catabolic processes occurring in the explant during the

136

culture period . Thus, conclusions about catabolism could stil l be inferred from

comparisons of the final amino sugar content and the relative rate of proteoglycan

synthesis of each treatment group to those of the control group .

An amino sugar assay was chosen to measure relative changes in the proteoglycan

content of cultured explants of equine articular cartilage primarily because both of the

major types of glycosaminoglycan present have equal proportions . Contribution of

amino sugars from the hyaluronic acid and glycoprotein fractions of articular cartilage

was thought l ikely to be insignificant in comparison to the amounts released from the

proteoglycans. The use of an amino sugar assay also allowed for the use of a total

digestion of each explant. Total digestion of the explants was considered preferable

to the use of proteoglycan extraction techniques which leave a variable proportion of

the proteoglycan content of each explant behind.

The use of uronic acid assays, and to some extent the use of cationic dyes , neglects the

contribution of the keratansulphate component of equine articular cartilage . The ratio

of chondroitin sulphate to keratansulphate is not consistent between horses , joints, sites

within a joint, or even with distance from the articular surface . Furthermore,

keratansulphate side-chains tend to be concentrated on the hyaluronic acid binding end

of proteoglycan molecules while chondroitin sulphate predominates on the distal two

thirds . Proteolytic cleavage part of the way along a proteoglycan molecule could

possibly reduce the proportion of chondroitin sulphate relative to the proportion of

keratansulphate remaining restrained within cultured explants of articular cartilage.

Cationic dyes can also be affected by the type and length of the glycosaminoglycans

remaining bound to the hyaluronic acid molecule.

The determination of individual amino sugar concentrations by ion chromatographic

methods may have been more precise than a non-specific amino sugar assay (Vachon

et al. , 1990), but the availability and expense of this technology precluded its use for

these experiments . Although time consuming, use of the Gatt & Berman (1966) amino

sugar assay required minimal sample manipulation and was relatively simple to

perform. The absorbances produced by galactosamine and glucosamine at 524 run

- 137

were not identical, but the differences were substantially smaller than those seen with

the use of dimethylmethylene blue assays. Furthermore, interference in the assay by \

other components of equine articular cartilage was shown not to be a major concern

(see Materials & Methods: Section One).

Incorporation of 35S has commonly been used for comparing rates of proteoglycan

synthesis between explants of articular cartilage. On average, both chondroitin

sulphate and keratansulphate contain one sulphate group per disaccharide unit. The

addition of the sulphate group occurs relatively late in the synthesis of these

glycosaminoglycans and accounts for most of the sulphate fiXation occurring in

articular cartilage. The sulphate groups attached to chondroitin sulphate and keratan

sulphate, with their associated negative charges, are responsible for much of the

physicochemical and resulting biomechanical properties of articular cartilage .

The use of cationic dyes such as safranin-0 and alcian blue for quantitative histological

comparisons of proteoglycan content, and or distribution changes , is subject to poor

repeatability (Hunziker et al. , 1992) . Staining intensities can be affected by variable

proteoglycan losses occurring during routine fixation and processing, as well as by the

types and lengths of the glycosaminoglycan side-chains remaining bound to the protein

cores of the proteoglycan molecules. Ideally, large numbers of explants would need

to be fixed in the presence of a cationic dye such as ruthenium then processed, cut, and

stained as a single batch to allow even semi-quantitative comparisons to be made

(Hunziker et al. , 1 992) . For these reasons very limited conclusions were drawn about

changes in proteoglycan content from the histological study .

The use of wet weights to standardise the cellular and proteoglycan content of explants

precluded use of part of the same explant for histological sectioning. Explants were

thus used from a different site in the same joints, and interpretations of the combined

results assumed explants from both sites behaved in a similar manner. The histological

results confirmed much of the variability shown to exist between the explants by the

biochemical analyses. Cell numbers varied greatly between sections as did the

appearance of the individual chondrocytes in the various zones. Some of this variation

138

was due to differences in sampling depths, but most was caused by inherent variation

in cellular distributions and characteristics known to occur within even small areas of

a joint. It was concluded that a great deal of care must be exercised in interpreting

differences between histological sections, even from the same site let alone from

different joints and horses . Expressing dead cell numbers as percentages of the total

number of nucleated cells removed some of the variation caused by the greatly

differing cell numbers in the explants . The percentage of chondrocytes found dead in

each explant was then reduced by the average percentage of chondrocytes found dead

in the non-cultured group of explants from the same horse. This was an attempt to

exclude that proportion of chondrocytes whose death may have been associated with

variations in processing times , and was probably not due to either culture or drug

effects . When the individual results were combined into their respective

control/treatment groups the distribution of results within most of the groups tended to

be skewed towards zero . However, a log transformation normalised the distributions

and standardised the variances of the different groups.

Histological assessment of nuclear morphology was considered the most objective and

definitive measure of the cytotoxic affects of different drug amounts/concentrations .

In the preliminary studies substantial variation was seen in both the cytoplasmic and

nuclear staining characteristics of sections cut from similarly treated explants.

Classification of cell viability purely on the nuclear morphology at 10,000 x

magnification produced the most definitive results . Exclusion of anuclear cells from

both the total and dead cell counts may have underestimated dead cell percentages , but

was used because there was no way of determining what percentage of the 'empty

lacunae' were due to sectioning orientation or artefact. Degenerative nuclear changes

have been used by other researchers as an assessment of cytotoxicity (Shoemaker et al. , 1 992; Gustafson et al. , 1992) .

Explant culture provided a closed system in which interactions between drug and tissue

could be studied under controlled conditions which involved minimal changes to either

cellular phenotypic expression or drug-matrix interactions. Explants remained

metabolically active and viable, but there was a net loss of proteoglycans during

139

culture . One of the initial aims of using an in vitro model was to assess what effects

the drugs to be tested had on 'normal ' articular cartilage. However t it was obvious

from preliminary experimental work that the assumption of normality could not be

applied further than the source of the tissue. The metabolic behaviour of the explants

more closely resembled the in vivo response of articular cartilage to injury . During

culture there were reductions in both cell viability and proteoglycan content, while the

rate of proteoglycan synthesis increased markedly . This was so especially for a period

immediately following culture initiation, but thereafter rates of proteoglycan synthesis

and catabolism were more constant, although still resulting in a net depletion of

proteoglycans from the explants .

B. DISCUSSION OF THE RESULTS

140

Methylprednisolone acetate (MPA) and phenylbutazone (PBZ) are the most common

steroid and non-steroidal anti-inflammatory drugs used for treatment of joint injury in

equine athletes. Their soft tissue mediated clinical effects are well recognised .

Whether or not they also confer some degree of chondroprotection is actively debated

(Tobin et al. , 1986; Mcllwraith & Vachon, 1988) . Furthermore , there is some

evidence to suggest their use may actually potentiate the progression of joint

deterioration (Gabel, 1978; Owen, 1980; Chunekamrai et al. , 1 989; Trotter et al. ,

199 1 ) . Both the types and mechanisms of their effects on articular cartilage are

subjects of some conjecture and much controversy. Despite the controversy, relatively

few controlled in vivo trials have investigated the effects of MPA or PBZ on equine

articular cartilage . Interpretation of these trials has been hindered by small sample

numbers, a wide range of confounding variables, and the limited number and type of

investigative procedures performed .

Several investigations have attempted to reduce the variation in composition between

explants by analysing large groups together. As a consequence most of the

experiments have been restricted to very small sample sizes. In the present experiment

inherent within-horse and between-horse variation in proteoglycan and cellular content

was reduced by dividing the results of each explant by its initial wet weight, then

further dividing these results by the mean of the control group from the same horse .

In this way sample numbers were maximised while some of the inherent variation was

controlled . The statistical power of each comparison was further increased by

analysing the results using the method of linear contrasts, which allowed examination

of the three major hypotheses by nonorthogonal combinations of the treatment and

control groups . However, lack of statistical power caused by the large inherent

variation between explants was still a major limitation to the extent to which the amino

sugar content results could be analyzed and interpreted .

For each explant, amino sugar content was used as a measure of proteoglycan content

(Glade et al. , 1 983 ; Richardson & Clark, 1 990; Vachon et al. , 1991) , 35S incorporation

141

as a measure of the rate of proteoglycan synthesis (Maroudas & Evans, 1 974; Tyler,

1 985 ; Chunekamrai et al. , 1989), and the number of pyknotic nuclei as a measure of

cell death (Ham, 1974; Mcllwraith & Van Sickle, 198 1 ; Shoemaker et al. , 1 992). The

two lowest drug concentrations were chosen to encompass the range likely to be present

in equine synovial fluid following the administration of clinically recommended doses

(Lehmann et al. , 1 98 1 ; Autefage et al. , 1986; Lees et al. , 1987) . However, the

extent to which the methylprednisolone acetate (MP A) doses dissolved in the culture

media, or were hydrolysed to the pharmacologically active methylprednisolone (MP)

form, were unknown.

A commercial formulation of MPA was used , as opposed to MPA or MP alone , to

ensure the results more closely approximated the clinical scenario. The extents to

which the MPA doses dissolved in the culture media, or were hydrolysed to the

pharmacologically active MP form by the serum colinesterases , were unknown. No

attempt was made to control for any effects the excipients of Depo-Medrol may have

had on the experiments. Accordingly , the conclusions drawn are restricted to

statements about the effect of Depo-Medrol on cultured explants of equine carpal

articular cartilage, although the possible mechanisms of action are discussed.

The 35S scintillation results were more variable than the amino sugar content results but

differences between treatment groups were also expected to be larger. Statistical power

was thus not a limiting factor in the analysis of these results, but for the sake of

interpretation both sets of results were analyzed in a similar fashion. To facilitate this

a number of different transformations were tried in an attempt to normalise the sample

population distributions of the different groups, and to standardise their variances .

None o f these transformations resulted i n distributions which satisfied Bartletts test for

equal variances (see Appendix}, but a square root transformation resulted in the lowest

X2 value for Bartlett' s test. The variances associated with the 2000 J.Lg mL-1 treatment

groups of both drugs were the only ones markedly different from the others. For

parametric analyses, the assumption of equal variances is most critical when sample

sizes are small or markedly different (Sokal & Rohlf, 198 1 ) . The size of the samples

and the differences between the groups were both large enough that we did not consider

142

the violation of equal variances would significantly affect the results at the level they

were to be interpreted.

The presence of Depo-Medrol in the culture media caused a dose related suppression

of proteoglycan synthesis in the explants . The permanence of this suppression was not

assessed. The suppressive effect of Depo-Medrol was significant at clinically

applicable doses of MPA (20-200 JLg mL-1), and was significantly greater at the 2000

JLg mL-1 dose . Paradoxically, the presence of Depo-Medrol in the culture media

reduced the depletion of proteoglycans from the explants . There was limited evidence

(p = 0.075) to suggest this effect was present at the clinically applicable doses,

supported by the fact that there was less evidence to suggest the effect was any greater

at the 2000 JLg mL-1 dose (p = 0. 135). Theoretically , proteoglycan depletion can arise

either from increase in catabolism or decrease in synthesis . Because rate of

proteoglycan synthesis appeared to have little influence on the final proteoglycan

content of the explants, it was concluded that any reductions in proteoglycan content

were mainly the result of catabolic processes . Furthermore , the positive correlation

between cell death and proteoglycan content suggested increases in proteoglycan

catabolism were predominantly due to metabolically active processes . Depo-Medrol

was cytotoxic at only the 2000 JLg mL-1 dose rate . Lysosomal enzyme release

associated with cell death would not seem to have been a major cause of proteoglycan

depletion.

The effect of Depo-Medrol on the synthesis of proteoglycans in equine articular

cartilage , as measured by radiolabel incorporation, has previously been reported by

only two studies (Chunekamrai et al. , 1989; Fubini et al. , 1 993) . Fubini et al. (1993)

reported that there was no significant difference between the rate of incorporation of

35S by articular cartilage from control joints versus that from joints treated with Depo­

Medrol 21 days previous. In contrast, Chunekamrai et al. ( 1989) found that following

8 weekly intra-articular administrations of Depo-Medrol, proteoglycan synthesis was

reversibly suppressed for periods in excess of 8 weeks. Similar fmdings have also

been reported in other species subjected to multiple and frequent administrations of

large doses of sparingly soluble steroid preparations (Mankin & Conger, 1966; Mankin

143

et al. , 1 972; Behrens et al. , 197 5). Generally, the degree and duration of suppression

has been proportional to the number, size, and frequency of doses.

The reduced proteoglycan depletion in this study is in agreement with the in vitro

fmdings of Jacoby (1976) and Tyler et al. (1982) , but not with those of Pool et al.

( 1 981 ) . However, the later were part of a preliminary report which gave few details

of the methods used. In vivo studies of MPA on the proteoglycan content of equine

articular cartilage (Chunekamrai et al. , 1 989; Trotter et al. , 199 1 ; Shoemaker et al. ,

1 992) have all reported that MPA adversely affected the cartilage proteoglycan content

of treated joints . These experiments involved multiple administrations of Depo­

Medrol, resulting in the articular cartilage of the treated joints being exposed to

relatively high concentrations of MPA \MP for extended periods. It is likely that under

these circumstances the suppressive effects of MP A \MP on cellular metabolism would

assume a greater relative importance over the anti-catabolic effects. This may explain

why our fmdings differ substantially from these in vivo trials

This is the first study to confirm that high doses of Depo-Medrol have a direct toxic

effect on equine chondrocytes. Interpretations of previous in vivo investigations

(Chunekamrai et al. , 1989; Shoemaker et al. , 1 992) have been complicated by

coincidental proteoglycan losses, which may have also left the chondrocytes more

susceptible to physical damage (Behrens et al. , 1976; Mcllwraith & Vachon, 1 988) .

Chondronecrosis, empty lacunae, intra-cartilaginous cysts, and deposition of

hydroxyapatite have all been reported following intra-articular depot corticosteroid

administrations in live animals (Behrens et al. , 1 976; Ohira & Ishikawa, 1986;

Chunekamrai et al. , 1989; Shoemaker et al. , 1992) . The cytotoxic mechanism of

action of corticosteroids for most tissues is thought to involve impairment of metabolic

function (Haynes, 1 991) . The almost total inhibition of proteoglycan synthesis caused

by the 2000 #Lg mL·1 dose of MP A, and the increased percentage of cells with smaller

nuclei and vacuolated cytoplasm, suggests this mechanism may also be important in

equine articular cartilage. In equine joints, an increased incidence of degenerative

cellular lesions has been associated with frequent administration of high doses of MP A

(Chunekamrai et al. , 1 989; Shoemaker et al. , 1992) . Investigations involving lower

144

doses of MP A, and/or longer dose intervals, have tended to show fewer or no changes

in chondrocyte viability (Marcoux, 1977; Trotter et al. , 199 1 ) . Thus, drug

concentration, duration of exposure, and physical factors probably all determine how

MPA affects chondrocyte viability in vivo.

Several in vivo studies have used the excipients of Depo-Medrol (water, Polyethylene

glycol 3350, myristyl-gamma-picolinium chloride (MGP)) as a treatment control and

showed no difference in either chondrocyte morphology or matrix composition from

the controls (Marcoux, 1978; Myers & Stack, 1 988; Chunekamrai et al. , 1 989) .

However, Myers & Stack (1988) did find one of the excipients (MGP) caused a dose­

dependant suppression of glycosaminoglycan synthesis when added at relatively high

concentrations to in vitro cultures of canine articular cartilage . It is therefore possible

that at the highest dose rate MGP may have also contributed to some of the suppressive

effects seen.

Because Depo-Medrol suppressed proteoglycan synthesis in all treatment groups it

cannot strictly be classified as a chondroprotective drug in the horse (as defined by

Burkhardt & Ghosh, 1 987) . However, in many clinical situations the benefits

associated with the anti-catabolic actions of Depo-Medrol could far outweigh the

suppressive effect on proteoglycan synthesis . Assuming the anti-anabolic effects are

not permanent (Fubini et al. , 1993) , the proteoglycan content of articular cartilage is

unlikely to be markedly affected by a single intra-articular administration of Depo­

Medrol. Multiple short interval administrations would result in the articular cartilage

within the joint being exposed to higher concentrations of MP A/MP for extended

periods. Under these circumstances the anti-anabolic , and possibly even the cytotoxic ,

effects could assume greater importance. Also the administration of Depo-Medrol in

joints with substantial pre-existing proteoglycan depletion, or cell death, might increase

the time taken by the chondrocytes to replenish the proteoglycan content of these joints .

Phenylbutazone and other NSAIDs have been reported to interfere with cellular

carbohydrate metabolism, and the synthesis of sulphated proteoglycans, in several

species (Whitehouse & Haslam, 1 962; Whitehouse, 1964; Bostrom et al. , 1964;

145

Palmoski & Brandt, 1 979; Palmoski et al. , 1980; Palmoski & Brandt, 1 983 ; Burkhardt

& Ghosh, 1987) . However, the literature is lacking in controlled reports of the effects \

of PBZ on equine articular cartilage . To our knowledge, this is the first reported

investigation of the effects of PBZ on cultured explants of equine articular cartilage .

The absence of any metabolic or cytotoxic effects at the lower concentrations (2-200

J.Lg ml-1) supports the in vivo findings of Denko ( 1964) (rat cartilage), but not the in

vitro results of Whitehouse & Bostrum (1962) and Bostrom et al. (1964) (bovine

cartilage) . It is possible species variations in enzyme structure and affinity for PBZ

may account for some of the differences.

Our results are consistent with PBZ being classified as a chondroprotective drug in the

horse . At clinically applicable concentrations PBZ significantly reduced proteoglycan

depletion, while having no effect on either proteoglycan synthesis or chondrocyte

viability . Metabolic and cytotoxic effects were seen only at the 2000 J..Lg ml-1

concentration, which is at least l OO-fold greater than concentrations achieved clinically

in equine synovial fluid (Lehmann et al. , 198 1 ; Lees et al. , 1987) . It was concluded

that the use of PBZ at clinically recommended dose rates was unlikely to impair

directly the proteoglycan or cellular content of equine joints, while conferring some

protective advantage against chondrocyte mediated catabolism.

This study demonstrates that the chondrocytes are quite capable of causing a substantial

depletion of proteoglycans by themselves without any help from cytokines or

proteolytic enzymes produced by synovial and migrant inflammatory cells. The

increased proteoglycan catabolism may have resulted from increased enzyme synthesis ,

or activation of the substantial pool of latent enzymes already present in the matrix

(W oessner & Selzer, 1984; Martel-Pelletier et al. , 1985 ; Flannery et al. , 1992) .

However, as the molecular mechanisms involved in inducing the synthesis and

activation of proteolytic enzymes within articular cartilage are not fully understood

(Flannery et al. , 1992; MacDonald et al. , 1992) , we can only speculate about the

means by which these two drugs reduced proteoglycan catabolism.

146

Most of the clinical effects of PBZ to date have been explained by its inhibition of the

cyclo-oxygenase mediated production of prostaglandins (Vane, 1 976; Lees & Higgins,

1 985) . However, at high concentrations, PBZ can also inhibit other enzymes including

at least one matrix proteolytic enzyme (Whitehouse, 1 962; Bostrum et al. , 1 964;

Burkhardt & Ghosh, 1987; May et al. , 1988; Meschter et al. , 1990) . Equine

chondrocytes subjected to adverse culture conditions have been shown to produce

increased amounts of prostaglandin � and the matrix proteolytic enzyme stromelysin

(May et al. , 1991 ) . The effects of increased prostaglandin production by equine

chondrocytes are not known, but the addition of prostaglandins of the E series to

articular cartilage cultures of other species has been shown to cause a proteoglycan

depletion (Fulkerson et al. , 1 979) . Prostaglandins can affect both the rate of synthesis

of new matrix proteolytic enzymes and the activation of the pool of latent enzymes

already present in the matrix (Burkhardt & Ghosh, 1 987) . Thus, it is likely the

chondroprotective effects of PBZ, like most of the clinical effects, may also

predominantly result from inhibition of prostaglandin production.

Corticosteroids generally exert their effects through 'transcriptional regulation' resulting

in an increased or decreased production of polypeptides (Haynes, 199 1 ) . These

polypeptides can be enzymes, enzyme potentiators, enzyme inhibitors , or have one of

many other effects on processes within the cel l . Corticosteroids can also inhibit the

production of inducible cyclo-oxygenase (COX-2) (Xie et al. , 1991 ; Kujubu et al. , 199 1 ; O'Banion et al. , 1 991) . Increased synthesis of COX-2 is responsible for much

of the sustained increases in prostaglandin production seen in many adversely

stimulated tissues (Simmons et al. , 1993 ; Winn et al. , 1 993) . Inhibition of

prostaglandin and or cytokine production by the chondrocytes may have been an

important mechanism of action involved in the anti-catabolic effect of Depo-Medrol .

Thus , the anticatabolic mechanism of action of Depo-Medrol could have involved either

direct of indirect suppression of new enzyme synthesis, or may have mainly involved

inhibition of the activation of proteolytic enzymes already present in the matrix .

However, as articular cartilage is thought to already contain a substantial pool of latent

proteolytic enzymes (Flannery et al. , 1992} , it is likely that much of the anti-catabolic

147

effect of Depo-Medrol resulted from a inhibition of the processes involved in the

activation of these enzymes, rather than just a suppression of their synthesis. Inhibition \ '

of prostaglandin production could conceivably have been a common mechanism of

action for both PBZ and Depo-Medrol . Further investigation is needed to defme what

mechanisms of action are involved in the anti-catabolic effects of these two drugs.

The histological observations generally agreed with the biochemical fmding that the

control group of explants for each drug was the most depleted in proteoglycan content.

The large variability in cellular and matrix staining characteristics between explants

further highlights the need for very large sample numbers for histological methods to

demonstrate treatment differences reliably (Mankin, 1 97 1 ; Hunziker et al. , 1992) .

Assessment of cell viability solely on nuclear morphology provided the most

unequivocal measure of cell death. With the exception of the areas immediately

adjacent to the cut edges of the explants, the distributions of proteoglycan loss and

chondrocyte death were similar to those found in in vivo models of inflammatory joint

disease (Mcllwraith & Van Sickle, 198 1 ; Gustafson et al. , 1992) .

The small number of chondrocyte clusters seen in any one section supports the finding

of others that the mitotic response of chondrocytes to insult is very limited (Hurtig et

al. , 1988; Richardson & Clark, 1 991) . In contrast to the finding of Shoemaker et al.

(1992) Depo-Medrol did not noticeably stimulate chondrocyte cluster formation. The

appearance of the chondrocyte clusters found in the transitional and radiate zones is

consistent with the finding of Poole et al. ( 1988) that the chondrocytes of these zones

exist within fibrous capsules. The constraint of chondrocytes within felt-like

pericellular capsules may help explain why damaged areas of articular cartilage are

rarely repopulated, even though post-mitotic clusters of chondrocytes are often seen at

their margins.

C. SUMMARY & CONCLUSIONS

148

Methods were set up whereby explants of equine carpal articular cartilage could be

successfully cultured for periods in excess of five days . The metabolic behaviour of

the cultured explants resembled the in vivo response of articular cartilage to injury .

All explants remained metabolically active and viable but suffered a net loss of

proteoglycans. Proteoglycan loss was reduced by the presence of either

phenylbutazone or Depo-Medrol . Depo-Medrol caused a dose-dependent suppression

of proteoglycan synthesis at all concentrations, but chondrocyte viability was markedly

inhibited only at doses well in excess of those used clinically. Phenylbutazone affected

proteoglycan synthesis and cell viability at the very highest concentrations used only .

At all concentrations, the anti-catabolic effects of each drug influenced the proteoglycan

content of the explants far more than did any anti-anabolic or cytotoxic drug effect.

The results suggest that the therapeutic potential of both phenylbutazone and Depo­

Medrol may not be just restricted to their anti-inflammatory effects on the soft tissues

of the joint, but may also involve a suppression of the synthesis and/or activation of

proteolytic enzymes within the cartilage itself. Furthermore, the results cast some

doubt over whether clinically recommended doses of either of these drugs substantially

contribute to the progression of degenerative joint disease through any direct negative

effect on the chondrocytes .

However, the extrapolation of in vitro results to clinical situations has always been

somewhat tenuous, especially when only a limited number of parameters are measured .

This study examined only quantitative differences in the proteoglycan content of the

matrix of cultured explants of equine articular cartilage. Changes in the functional

capacity of the restraining collagen network were not looked for, nor were any

qualitative changes in the proteoglycans themselves. Never-the-less, in vitro studies

can improve the understanding of the mechanisms of drug action and their relationship

to concentration. This information, when combined with an understanding of the

pathogenesis of the condition being treated, can lead to more rational drug use.

149

The clinical application of these results is also limited because the study examined only

the short term, dose related effects of a MP A formulation on chondrocytes, isolated

from othe; physiological and physical influences. No attempt was made to determine

how much of the MPA was converted into the active MP form. Continued

investigation is required to assess the concentration related effects of MP alone, and

to determine the duration of its suppressive effects on proteoglycan synthesis .

Cartilage destruction associated with inflammatory joint disease may also involve

chondrocytes being influenced by cytokines produced by other cell types, as well as

proteolytic enzymes released from the synoviocytes and migrant inflammatory cells .

Further research is needed to define what effects

(a) IL- l , TNF, and PG� have on proteoglycan synthesis and catabolism.

(b) MP and PBZ may have on the effects of IL- l , TNF, and PG�.

(c) MP and PBZ have on the release of cytokines and proteolytic enzymes

from cultures of equine synovium, neutrophils and monocytes.

Future investigations should concentrate on clinically applicable concentrations of MP,

PBZ, and cytokines and perhaps could also investigate whether combinations of MP

and PBZ may have an additive or synergistic effect.

150

APPENDIX

A. TABLES \

p.g Galactosamine Glucosamine Chondroitin SO 4 2 .5 0 .07 1 0.079 0 .072

5 0. 139 0 . 149 0 . 1 23

10 0 .262 0.287 0 .2 19

15 0. 398 0 .420 0 .331

20 0.532 0.540 0.432

Table 1 . Mean absorbances at 524 nm of three sets of standard solutions assayed for their amino sugar content.

p.g Amino sugar 0.5 hours 2 hours 23 hours

Glucosamine

5 0. 146 0. 1 3 1 0 .090

10 0.266 0.239 0. 162

15 0.41 8 0. 377 0 . 243

20 0. 554 0.503 0 . 3 1 5

Galactosamine

5 0. 1 5 1 0. 1 30 0 .076

10 0. 284 0.255 0 . 156

15 0 .391 0.348 0 . 2 1 9

20 0 .5 1 6 0.462 0 . 274

Chondroitin sulphate

5 0 . 1 19 0 . 1 07 0 .068

1 0 0 . 2 1 8 0.202 0 . 1 24

1 5 0.295 0.272 0 . 1 65

20 0.392 0.362 0 . 2 1 0

Table 2. Effect of time on the amino sugar assay (absorbances at 524 run)

151

Standard cpm dpm CE H number

A

B

c D

E

F

Table 3 .

Table 4 .

Table 5 .

191 3 1 2 201770

1 83871 201770

1 6705 1 201770

1463 1 6 201770

1 33324 201 770

1 10333 201 770

14C Quench standards

Washes

0

2

3

Live chondrocytes

1 0653 ± 4525

3938 ± 1072

3582 ± 1033

0 .948 57

0 .9 1 1 1 28

0 .828 2 14

0 .725 282

0 .66 1 3 12

0. 547 350

Dead chondrocytes

6393 ± 147 1

162 ± 1 72

80 ± 1 00

Effect of two different wash techniques on biologically bound and non-bound 358 content. (mean dpm mg-1)

DNA Rep 1 Rep 2 Rep 3 Mean

(ng) (fluorescence)

Blank 0.00 0.01 0 .02 0.01

100 0. 1 3 0. 14 0 . 1 6 0.14

200 0 .30 0.30 0 .30 0.30

300 0 .47 0.46 0 . 44 0.46

400 0 .61 0.60 0 . 55 0.59

500 0 . 77 0 .71 0 .70 0.73

Assay of calf thymus DNA standards (ng DNA versus fluorescence) .

152

Galactosamine (p.g) Rep 1 Rep 2 Mean

Table 6.

3.75

7.50

15.0

22.5

0.050

0. 135

0.230

0.360

0.060

0. 140

0 .265

0. 385

0.055

0. 138

0.248

0.373

Absorbances from chondroitin sulphate standards in water, dialysed and oven digested .

Galactosamine (p.g) Rep 1 Rep 2 Mean

3.75 0.040 0 .080 0.060

7.50 0. 105 0. 160 0. 133

15.0 0. 1 70 0. 220 0. 195

22.5 0.415 0 .415 0.410

Table 7. Absorbances from chondroitin sulphate standards in DMEM, dialysed and oven digested.

Day Explants Mean AS Mean Weight AS Weight-1 ± SD CV (n) (p.g) (mg) (p.g mg-1) (%)

0 10 79 .8 3 . 59 22 . 5 ± 5 . 4 24

1 10 75 . 8 4 . 1 1 1 8 . 2 ± 3 . 7 20

2 10 76.2 4 . 1 2 1 8 . 4 ± 4 . 1 22

3 10 58 . 8 3 . 7 1 1 5 . 8 ± 1 .9 1 2

4 10 52 .6 3 . 8 1 1 3 . 8 ± 3 . 3 24

5 10 63 . 4 3 . 82 1 6 . 5 ± 2 . 6 1 6

6 9 6 1 .7 3 . 74 16 .5 ± 2 .4 14

7 10 55 .0 3 .53 1 5 . 5 ± 3 . 3 2 1

mean 3.80 19

Table 8. Effect of culture duration on the amino sugar content (AS) of cartilage explants relative to their initial wet weight.

Day

0

1

2

3

4

5

6

7

mean

Table 9.

Day

0

1

2

3

4

5

mean

Table 10.

153

Explants Mean AS Mean DNA AS DNA·1 ± SD CV (n) (JLg) (JLg) (JLg JLg-1) (%)

1 0 79. 8 2 . 7 1 29.7 ± 5 .9 20

10 75 .8 3 . 08 24. 8 ± 7 . 2 29

10 76 .2 2 . 82 26.9 ± 7 . 8 29

1 0 58.8 2 .92 20.4 ± 4.3 21

10 52 .6 2 . 14 25 .5 ± 8 . 7 34

10 63 . 4 2 .21 28. 8 ± 5 . 8 20

9 6 1 .7 2 . 54 24 .4 ± 5 .4 22

10 55 . 0 2 . 1 8 26 .0 ± 8 . 8 35

2.58 26

Effect of culture duration on the amino sugar content (AS) of cartilage explants relative to their final DNA content.

Explants Mean dpm Mean Weight dpm Weight·1 ± SD CV (n) (mg) (dpm mg-1) (%)

10 21 3 .59 6 ± 5 83

1 0 2 1 59 4 . 1 1 525 ± 176 34

1 0 4414 4 . 12 1074 ± 345 32

1 0 5655 3 .71 1520 ± 796 52

1 0 5384 3 . 81 1466 ± 786 54

1 0 55 1 5 3 . 82 1435 ± 5 12 36

3.86 * 42

* mean excludes day 0 C. V. %

Effect of culture duration on the incorporation of 35S by cartilage explants relative to their initial wet weight.

154

Day Explants Mean dpm Mean DNA dpm DNA-1 ± SD CV (�tg) (dpm ILg-1) (%)

(n)

0 1 0 2 1 2 . 7 7 ± 6 76

1 1 0 2159 3 . 1 700 ± 260 37

2 1 0 44 14 2 .8 1 552 ± 45 1 29

3 ' 10 5655 2 . 9 1 860 ± 879 47

4 1 0 5384 2 . 1 2546 ± 1058 42

5 1 0 55 15 2 .2 2529 ± 1016 40

mean 2.6 * 39

* mean excludes day 0 C. V. %

Table 1 1 . Effect of culture duration on the incorporation o f 358 by cartilage explants relative to their final DNA content.

155

Treatment Explants Mean % control group (n) ± SEM •

Control 61 A 100.00 ± 3 .28

20 p.g mL·1 67 8 108. 12 ± 3.04

200 p.g mL·1 69 c 106.54 ± 3.36

2000 p.g mL·1 66 D 113.31 ± 2.32

Table 12.

•

The dose related effect of a methylprednisolone acetate formulation on the amino sugar content of cultured explants of equine middle carpal joint articular cartilage.

The amino sugar mg·1 value of each explant was divided by the mean amino sugar mg·1 value of the control group from the same horse . All results were then combined into four control/treatment groups, averaged and expressed as percentages.

When B, C, and D are combined they are significantly different from A (p = 0.017). When just B and C are combined they are not significantly different from A (p = 0.075). D is not significantly different from groups B and C combined .

156

Treatment Explants Mean % control Square root group (n) ± SEM • transformation ± SEM • •

Control 61 99.99 ± 4.92 A 0.9836 ± 0.2320

20 J!g mL·1 67 76.15 ± 3.64 B 0.8543 ± 0.0219

200 J!g mL·1 69 50.06 ± 3.42 c 0.6800 ± 0.0237

2000 J!g mL·1 66 5.54 ± 0.85 D 0.2035 ± 0.0151

Table 13. The dose related effect of a methylprednisolone acetate formulation on the incorporation of 358 by cultured explants of equine middle carpal joint articular cartilage.

The dpm mg·1 value of each explant was divided by the mean dpm mg·1 value of the control group from the same horse. All results were then combined into four control/treatment groups, averaged and expressed as percentages. The dpm mg·1 value of each explant was divided by the mean dpm mg·1 value of the control group from the same horse. The square root of each result was then calculated before all the results were combined into the four control/treatment groups and averaged .

When B, C, and D are combined they are significantly different from A (p < 0.001) . Group A is also significantly different from groups B and C combined (p < 0.001) . Groups B and C combined are significantly different from group D (p < 0. 00 1 ) .

157

Treatment Ex plants Cells(%) Cells(%) dying Log (Cells(%) dyinl group (n) dead during culture during culture + l

Mean ± SD · Mean ± SD Mean ± SD

Not cultured 35 1 . 1 ± 1 .3

Control 35 2.9 ± 2.7 1 .9 ± 2.2 0.360 ± 0.299

20 p.g mL-1 36 1 .9 ± 1 .4 1 . 1 ± 1.3 0.255 ± 0.238

200 p.g mL-1 36 2.9 ± 3.0 1 .9 ± 2.2 0.356 ± 0.298

2000 p.g mL-1 36 12.6 ± 9.4 1 1 .5 ± 9.0 0.960 ± 0.385

Table 14. The dose related effect of a methylprednisolone acetate formulation on the viability of chondrocytes within cultured explants of equine middle carpal joint articular cartilage.

The percentage of chondrocytes determined as dead in each cultured explant was reduced by the average percentage of chondrocytes found dead in the non-cultured group of ex plants from the same horse . Any resulting negative percentages were rounded up to zero, then all results were combined into the four control/treatment groups and averaged .

The result from the 2000 p.g mL-1 group was the only one significantly different from the others (p < 0. 001) .

158

Treatment Explants Mean % of control group (n;) ± SEM "'

Control 62 A 100.00 ± 2 .92

2 p.g mL·1 61 B 106.46 ± 2.81

20 p.g mL·1 62 c 1 1 1 .32 ± 3.95

200 p.g mL·1 60 D 108.67 ± 3 .75

2000 p.g mL·1 36 E 113.35 ± 4.79

Table 15.

"'

The effect of different concentrations of phenylbutazone on the amino sugar content of cultured explants of equine middle carpal joint articular cartilage.

The amino sugar mg·' value of each explant was divided by the mean amino sugar mg·' value of the control group from the same horse _ All results were then combined into five control/treatment groups, averaged and expressed as percentages.

Groups B, C, D, and E combined are significantly different from group A (p = 0.01 1 ) . A is also significantly different from groups B and C combined (p < 0.00 1 ) . Groups B and C combined are not significantly different from groups D and E combined (p = 0 .59).

159

Treatment Explants Mean % control Square root transformation group (n) ± SEM * ± SEM • •

Control 62 99.99 ± 3.60 0.9905 ± 0.0176

2 p.g mL-1 61 103.09 ± 3 .18 1 .0080 ± 0.0157

20 p.g mL-1 62 92.54 ± 3.02 0.9536 ± 0.0160

200 p.g mL-1 60 98.29 ± 3.32 0.9826 ± 0.0172

2000 p.g mL-1 36 5.13 ± 0 .14 0.0057 ± 0.0073

Table 16.

*

* *

The effect of different concentrations of PBZ on the incorporation of 358 by cultured explants of equine middle carpal joint articular cartilage.

The dpm mg-1 value of each explant was divided by the mean dpm mg-1 value of the control group from the same horse. All results were then combined into five control/treatment groups, averaged and expressed as percentages . The dpm mg-1 value of each explant was divided by the mean dpm mg-1 value of the control group from the same horse . The square root of each result was then calculated before all the results were combined into the five control/treatment groups and averaged .

The result from the 2000 p.g mL-1 group was the only one significantly different from the others (p < 0.001) .

160

Treatment Explants Cells(%) Cells(%) dying Log (Cells(%) dying group (n) dead during culture during culture + 1)

Mean ± SD Mean ± SD •••• Mean ± SD

Not cultured 29 . 1 .0 ± 1 . 1

Control 32 2.2 ± 2.4 1 .6 ± 2.2 0.302 ± 0.295

2 p.g mL·1 32 2.9 ± 3 . 1 2.3 ± 3 . 1 0.393 ± 0.324

20 p.g mL·1 32 2 .1 ± 1 .9 1 .5 ± 1 .9 0.316 ± 0.262

200 p.g mL·1 31 •• 1 .8 ± 2 . 1 1 .3 ± 2 .3 0.251 ± 0.288

2000 p.g mL"1 28 ••• 9.8 ± 4.8 9.8 ± 5 . 1 0.913 ± 0.336

Table 17. The effect of different concentrations of phenylbutazone on the viability of chondrocytes within cultured explants of equine middle carpal joint articular cartilage.

Non-cultured ex plants were not kept from one horse, and four out of the five other horses had six explants kept. One explant went missing during processing . Only four explants instead of five were cultured from four of the six horses. The percentage of chondrocytes determined as dead in each cultured explant was reduced by the average percentage of chondrocytes found dead in the non-cultured group of explants from the same horse . Any resulting negative percentages were rounded up to zero , then all results were combined into the five control/treatment groups and averaged .

The result from the 2000 1-Lg mL-1 was the only one significantly different from the others (p < 0 . 001 ) .

B. STATISTICAL ANALYSES

Method of analysis = Linear contrasts

Depo-Medrol trial

1 . Effect of Depo-Medrol on amino sugar content

First null hypothesis: Ucontror = Utreatrnents (Ui = Population mean)

Contrast = C = ct Y control + � Y 2011g mL-t + c3 Y 2oo,.g mL-t + C4 Y 2ooo,.g mL-t

Where Eci = 0

Yi = sample mean of the control or treatment groups

Relevant data from analysis of variance

p = 0 .0455

MSerror = 0 .07055

Bartletts X2 = 1 .26, p = 0 .7386

Ycontrol

y20 p.g mL-1 Y2oo p.g mL-t y 2000 p.g mL-1

sscontrast =

= 1 .0000, n1 = 61

= 1 .08 12, n2 = 67

= 1 .0654, n3 = 69

= 1 . 133 1 ' � = 66

= -0.2797

__cQ:_ - (-0.2797)2

E i£J: .W.: + ill + ill + ill

= 0 .4072

6 1 67 69 66

F ratio = SScrror = 0.4072 = 5 • 772 MSerror 0 . 07055

F(l, 259> , p = 0.01699

161

Second null hypothesis: V control = !6,.8 mL-1 + U200,.8 mL-1 2

Contrast

ss contrast F ratio

= = 0.1466

= = =

0 .2262

3 . 207

0.0749

Third null hypothesis: = !6,.8 mL-1 + U20011g mL-1 = Uzooo,.8 mL-1 2

Contrast

ss contrast

Fratio Fu,2oo>' P

= = 0.1 196

= = =

0. 1589

2 .2522

0. 1350

2 . Effect of Depo-Medrol on 35S04 incorporation

Relevant data from analysis of variance

(a) For raw data

p = 0 .0000

MSerror = 0.07889

Bartletts X2 = 1 36 .04, p = 0 .0000

Y control = 0 . 9999, n1 = 6 1

Y2o,.g mL-1 = 0 .7615 , n2 = 67

y200pg mL-1 = 0.5006, n3 = 69

y 2000pg mL-1 = 0.0554, � = 66 (n-k) = 259

162

(b) For square root transformation (sqrt) data

p = 0 .0000

MSerror = 0.02977

Bartletts X2 = 1 5 .25 , p = 0.0016

Y control = 0.9836, n1 = 6 1

Y2o,.g mL-1 = 0. 8543 , n2 = 67

y 200,.g mL-1 = 0. 6800, n3 = 69

y 2000,.g mL-1 = 0.2035 , 14 = 66

(c) For log transformed data

p = 0.0000

MSerror = 0. 10628

Bartletts X2 = 108.95 , p = 0.0000

Ycontrol = 3 .97 18 , n1 = 6 1

Y2o,g mL-1 = 3 . 8417, n2 = 67

Y2oo,g mL-1 = 3 .6247, n3 = 69

y 2000,.g mL-1 = 2 . 4672, 14 = 66

(n-k) = 259

(n-k) = 259

163

The equal variance assumption is most critical when sample sizes are markedly

different. The sqrt transformation results in the lowest X2 value for Bartletts test . The

variance associated with the high treatment group is primarily responsible for violating

the assumption of equal variances as its mean approaches zero. The differences

between the ·comparisons are so great that the violation of the assumption of equal

variances is highly unlikely to affect the results at the level they are to be interpreted .

First null hypothesis: Uamtrol = U,reatments

Using the sqrt transformed data

Contrast

ss contrast

Fstatistic

Fu,2s9)' P

-

-

-

=

-

4Y control + 1 Y2o"g mL-1 + 1Y2oo"g mL-I + 1Y2ooo"g mL-I

1 .213

7 . 66

257 . 4

0.000

Second null hypothesis Ucontrol = U2o"g mL- 1 + U2oo"g mL-1-

2

Contrast

ss contrast

Fo. 19Sl' P

=

= 0.4329

=

=

1 . 9728

66 .27

0.000

Third null hypothesis _!10"g mL-1 + U2oo"g mL-1- = U2ooo"g mL-1 2

Contrast

ss contrast

Fratio

Fo. 200)' P

=

=

=

=

1 . 1273

1 4 . 1 1 8

474 .25

0.0000

164

Phenylbutazone trial

1 . Effect of phenylbutazone on amino sugar content

First null hypothesis: Ucontrol = Utreatments

Relevant data from analysis of variance

(a) For raw data

p = 0 . 0933

MScrror = 0 . 07 123

Bartletts X2 = 1 0. 8 1 , p = 0.0288

Ycontrol = 1 .0000, n1 = 62

y2p.g mL-1 = 1 . 0646, n2 = 6 1

y20p.g mL-1 = 1 . 1 1 32, n3 = 62

y200p.g mL-1 = 1 . 0867 , 14 = 60

y 2000p.g mL-1 - 1 . 1 335 , n5 = 36 (n-k) = 276

Contrast = 4 Y control - Y 2p.g mL-1 - Y 20p.g mL-1 - Y 200p.g mL-1 - Y 2000p.g mL-1 - -0.3980

ss contrast = 0.4728 1

F ratio = 6 . 6377

Fu. 276)' P = 0.01050

165

(b) For square root transformation data

p = 0. 1 34 1

MSerror = 0.01722

Bartletts X2 = 9 . 59, p = 0.0478

Ycontrol

y21'g mL-1

y201'g mL-1

y2001'g mL-1

y 20001'g mL- 1

Contrast

ss contrast

Fratio

= 0 . 9933 , n1 = 62

= = = =

=

=

1 .0262, n2 = 6 1

1 . 0439, n3 = 62

1 . 033 1 , 14 = 60

1 . 0567, n5 = 36

-0.1867

0. 104

6 . 04

0.01460

(n-k) = 276

Second null hypothesis Ucontrol = U21'g m�.r1 + U201'g mL-1 2

(a) Non-transformed data

Contrast

ss contrast Fratio

F(l, 195>' P

----

-0. 1778

0.4880

6 . 846

0.00964

(b) Sqrt transformation data

Contrast - -0.0835

ss contrast

F ratio

F(l, 195) • P =

0.07 1 85

4 . 17

0.04210

166

Second null hypothesis = U2,.g mL-1 + U20,.g mL-1 = U200,.g mL-1 + U2000,.g mL-1

(a) Non-transformed data

Contrast

ss contrast

Fratio

Fu, 209>' P

= = = =

-0.0424

0.02336

0.3238

0.56995

(b) Sqrt transformation data

Contrast = -0.0197

ss contrast = 0.00504

Fratio = 0. 2928

Fu, 209> • P = 0.58902

2 . Effect of phenylbutazone on 35804 incorporation

First null hypothesis: Ucontrol = Utreatments

(a) For raw data

p = 0. 0000

MSerror = 0 . 05807

Bartletts X2 = 200. 84, p = 0. 0000

Ycontrol = 0 . 9999, n 1 = 62

Y2,.g mL-1 = 1 .0309, n2 = 6 1

y20pg mL-1 = 0 . 9254, n3 = 62

y200pg mL-1 = 0. 9829, 14 = 60 (n-k) = 276

y 2000pg mL-1 = 0 . 005 1 , n5 = 36

167

(b) For square root transformation data

p = 0. 0000

MSerror = 0 . 0 1 5 1 3

Bartletts X2 = 44 . 1 9, p = 0.0000

Y control = 0. 9905 , n1 = 62

y21'g mL-1 = 1 .0803 , n2 = 6 1

y201'g mL-1 = 0 . 9536 , n3 = 62

y2001'g mL-1 = 0. 9828, 14 = 60

y 20001'g mL-1 = 0.0572, n5 = 36

(c) For log transformed data

p = 0. 0000

MSerror = 0. 1 0628

Bartletts X2 = 108.95, p = 0. 0000

Y control = 3 . 9832, n1 = 62

y21'g mL-1 = 4 . 0005 , n2 = 61

y201'g mL-1 = 3 . 9508, n3 = 62

y2001'g mL-1 = 3 . 9765 , 14 = 60

y 20001'g mL-1 = 1 . 3306 , n5 = 36

(n-k) = 276

(n-k) = 276

168

NB: None of the transformations result in means with similar variances . The equal

variance assumption is most critical when sample sizes are markedly different . The

sqrt transformation results in the lowest X2 value for Bartletts test. The variance

associated with the extra treatment group, whose mean approaches zero, is primarily

responsible for violating the assumption of equal variances . Only the extra groups

mean is markedly different from the other means . The differences between the extra

groups mean and the other groups means are so great that the violation of the

assumption of equal variances is considered not to affect the results at the level they

are to be interpreted at.

Conclusion 1:

Conclusion 2:

Conclusion 3:

169

Phenylbutazone can effect the incorporation of radiolabel into

tissue cultured explants of equine articular cartilage.

The effect is not statistically significant at concentrations

achieved clinically in equine joints .

The effect is greatest at higher concentrations.

Note: These would be the conclusions reached if the data are analyzed in the same

way as the methylprednisolone data. However, examination of the data clearly

shows very little effect in any group other than the extra group, where the effect

is dramatic.

1 70

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